Search

Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications., Synopsis Candida albicans is a commonly encountered fungal pathogen usually responsible for superficial infections (thrush and vaginitis). However, an estimated 30% of severe fungal infections, most due to Candida, result in death. Those who are most at risk include individuals taking immune-suppressive drugs following organ transplantation, people with HIV infection, premature infants, and cancer patients undergoing chemotherapy. Current therapies for this pathogen are made more difficult by the significant secondary effects of anti-fungal drugs that target proteins that are also found in the human host. Recent sequencing and assembly of the genome for the fungal pathogen C. albicans used simple automated procedures for the identification of putative genes. Here, we report a detailed annotation of the 6,354 genes that are present in the genome sequence of this organism, essentially writing the dictionary of the C. albicans genome. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that are absent from the human genome and whose products might be targeted for antifungal therapy. The results of these efforts will thus ensure that the Candida research community has uniform and comprehensive genomic information for medical research, for the development of functional genomic tools as well as for future diagnostic and therapeutic applications.

Forward genetic screens have been used as a powerful strategy to dissect complex biological pathways in many model systems. A significant limitation of this approach has been the time-consuming and costly process of positional cloning and molecular characterization of the mutations isolated in these screens. Here, the authors describe a strategy using microarray hybridizations to facilitate positional cloning. This method relies on the fact that premature stop codons (i.e., nonsense mutations) constitute a frequent class of mutations isolated in screens and that nonsense mutant messenger RNAs are efficiently degraded by the conserved nonsense-mediated decay pathway. They validate this strategy by identifying two previously uncharacterized mutations: (1) tom-1, a mutation found in a forward genetic screen for enhanced acetylcholine secretion in Caenorhabditis elegans, and (2) an apparently spontaneous mutation in the hif-1 transcription factor gene. They further demonstrate the broad applicability of this strategy using other known mutants in C. elegans, Arabidopsis, and mouse. Characterization of tom-1 mutants suggests that TOM-1, the C. elegans ortholog of mammalian tomosyn, functions as an endogenous inhibitor of neurotransmitter secretion. These results also suggest that microarray hybridizations have the potential to significantly reduce the time and effort required for positional cloning., Synopsis Genetic screens are commonly used to figure out which genes are involved in a biological process. The first step in a genetic screen is to isolate mutant animals that are defective in the process being studied. The next step is to find which of the thousands of genes has the mutation that causes the observed defect. Positional cloning, the tried-and-true method for locating mutations, is slow and expensive. The authors propose using microarray hybridizations to speed the process. Their approach relies on the fact that a large fraction of the mutations found in screens are the results of premature stop codons, a particularly severe type of mutation. In cells, messages containing premature stop codons are rapidly destroyed by a protective pathway, called nonsense-mediated decay, thus making them directly detectable by microarray hybridization. The authors apply this strategy retrospectively to known mutants in Caenorhabditis elegans, Arabidopsis, and mouse. They identify two uncharacterized mutations in C. elegans, including one, tom-1, found in a forward genetic screen for enhancers of neurotransmission. Interestingly, their characterization of tom-1 mutants suggests that the highly conserved protein tomosyn inhibits neurotransmission in neurons. This study shows that microarray hybridizations will help reduce the time and effort required for positional cloning.

Although domestic cats (Felis silvestris catus) possess an otherwise functional sense of taste, they, unlike most mammals, do not prefer and may be unable to detect the sweetness of sugars. One possible explanation for this behavior is that cats lack the sensory system to taste sugars and therefore are indifferent to them. Drawing on work in mice, demonstrating that alleles of sweet-receptor genes predict low sugar intake, we examined the possibility that genes involved in the initial transduction of sweet perception might account for the indifference to sweet-tasting foods by cats. We characterized the sweet-receptor genes of domestic cats as well as those of other members of the Felidae family of obligate carnivores, tiger and cheetah. Because the mammalian sweet-taste receptor is formed by the dimerization of two proteins (T1R2 and T1R3; gene symbols Tas1r2 and Tas1r3), we identified and sequenced both genes in the cat by screening a feline genomic BAC library and by performing PCR with degenerate primers on cat genomic DNA. Gene expression was assessed by RT-PCR of taste tissue, in situ hybridization, and immunohistochemistry. The cat Tas1r3 gene shows high sequence similarity with functional Tas1r3 genes of other species. Message from Tas1r3 was detected by RT-PCR of taste tissue. In situ hybridization and immunohistochemical studies demonstrate that Tas1r3 is expressed, as expected, in taste buds. However, the cat Tas1r2 gene shows a 247-base pair microdeletion in exon 3 and stop codons in exons 4 and 6. There was no evidence of detectable mRNA from cat Tas1r2 by RT-PCR or in situ hybridization, and no evidence of protein expression by immunohistochemistry. Tas1r2 in tiger and cheetah and in six healthy adult domestic cats all show the similar deletion and stop codons. We conclude that cat Tas1r3 is an apparently functional and expressed receptor but that cat Tas1r2 is an unexpressed pseudogene. A functional sweet-taste receptor heteromer cannot form, and thus the cat lacks the receptor likely necessary for detection of sweet stimuli. This molecular change was very likely an important event in the evolution of the cat's carnivorous behavior., Synopsis Although sweet sugars are ubiquitous in human foods, they are seldom added to cat food, and owners usually do not feed sweets to their cats. This is because, in contrast to most other mammals, both domestic cats and their wild cousins, the big cats, do not show a preference for and, most likely, cannot detect sweet-tasting compounds. Other than this sweet blindness, the cat's sense of taste is normal. The molecular mechanism for this unique behavior towards sweets was not known, until now. Sweet compounds, including sugars and artificial sweeteners, are recognized by a special taste bud receptor composed of the products of two genes. The authors found that in cats, one of these genes is not functional and is not expressed. (It is called a pseudogene.) Because the sweet receptor cannot be formed, the cat cannot taste sweet stimuli. During the evolution of the cats' strictly carnivorous behavior, selection to maintain a functional receptor was apparently relaxed. This research provides a molecular explanation for the common observation that the cat lives in a different sensory world than the cat owner.

Plants are sessile and therefore have developed mechanisms to adapt to their environment, including the soil mineral nutrient composition. Ionomics is a developing functional genomic strategy designed to rapidly identify the genes and gene networks involved in regulating how plants acquire and accumulate these mineral nutrients from the soil. Here, we report on the coupling of high-throughput elemental profiling of shoot tissue from various Arabidopsis accessions with DNA microarray-based bulk segregant analysis and reverse genetics, for the rapid identification of genes from wild populations of Arabidopsis that are involved in regulating how plants acquire and accumulate Na+ from the soil. Elemental profiling of shoot tissue from 12 different Arabidopsis accessions revealed that two coastal populations of Arabidopsis collected from Tossa del Mar, Spain, and Tsu, Japan (Ts-1 and Tsu-1, respectively), accumulate higher shoot levels of Na+ than do Col-0 and other accessions. We identify AtHKT1, known to encode a Na+ transporter, as being the causal locus driving elevated shoot Na+ in both Ts-1 and Tsu-1. Furthermore, we establish that a deletion in a tandem repeat sequence approximately 5 kb upstream of AtHKT1 is responsible for the reduced root expression of AtHKT1 observed in these accessions. Reciprocal grafting experiments establish that this loss of AtHKT1 expression in roots is responsible for elevated shoot Na+. Interestingly, and in contrast to the hkt1–1 null mutant, under NaCl stress conditions, this novel AtHKT1 allele not only does not confer NaCl sensitivity but also cosegregates with elevated NaCl tolerance. We also present all our elemental profiling data in a new open access ionomics database, the Purdue Ionomics Information Management System (PiiMS; http://www.purdue.edu/dp/ionomics). Using DNA microarray-based genotyping has allowed us to rapidly identify AtHKT1 as the casual locus driving the natural variation in shoot Na+ accumulation we observed in Ts-1 and Tsu-1. Such an approach overcomes the limitations imposed by a lack of established genetic markers in most Arabidopsis accessions and opens up a vast and tractable source of natural variation for the identification of gene function not only in ionomics but also in many other biological processes., Synopsis Unlike most animals, plants are sessile and cannot leave a poor-quality environment after germinating. They therefore need to tolerate the particular conditions they encounter to survive. This makes plants an ideal system for the study of adaptive variation, and this is particularly true of Arabidopsis thaliana (Arabidopsis), which shows substantial natural variation and for which numerous genetic tools exist. Using a combination of analytical chemistry, genetics, and genomics, the authors were able to identify the specific genetic alteration that drive the natural variation in shoot sodium (Na+) accumulation capacity observed in Arabidopsis populations from coastal regions of Spain and Japan (Tossa del Mar and Tsu, respectively). They observed that a deletion in the DNA responsible for regulating the expression of HKT1, a gene known to encode for a Na+ transporter, causes reduced expression of AtHKT1 in roots of both the Spanish and Japanese populations. Such altered expression results in the elevated shoot Na+ observed in these two populations. Interestingly, this novel version of the HKT1 genes is also associated genetically with the enhanced NaCl resistance they observe in the Japanese population.

Many animals have a variety of pigment patterns, even within a species, and these patterns may be one of the driving forces of speciation. Recent molecular genetic studies on zebrafish have revealed that interaction among pigment cells plays a key role in pattern formation, but the mechanism of pattern formation is unclear. The zebrafish jaguar/obelix mutant has broader stripes than wild-type fish. In this mutant, the development of pigment cells is normal but their distribution is altered, making these fish ideal for studying the process of pigment pattern formation. Here, we utilized a positional cloning method to determine that the inwardly rectifying potassium channel 7.1 (Kir7.1) gene is responsible for pigment cell distribution among jaguar/obelix mutant fish. Furthermore, in jaguar/obelix mutant alleles, we identified amino acid changes in the conserved region of Kir7.1, each of which affected K+ channel activity as demonstrated by patch-clamp experiments. Injection of a bacterial artificial chromosome containing the wild-type Kir7.1 genomic sequence rescued the jaguar/obelix phenotype. From these results, we conclude that mutations in Kir7.1 are responsible for jaguar/obelix. We also determined that the ion channel function defect of melanophores expressing mutant Kir7.1 altered the cellular response to external signals. We discovered that mutant melanophores cannot respond correctly to the melanosome dispersion signal derived from the sympathetic neuron and that melanosome aggregation is constitutively activated. In zebrafish and medaka, it is well known that melanosome aggregation and subsequent melanophore death increase when fish are kept under constant light conditions. These observations indicate that melanophores of jaguar/obelix mutant fish have a defect in the signaling pathway downstream of the α2-adrenoceptor. Taken together, our results suggest that the cellular defect of the Kir7.1 mutation is directly responsible for the pattern change in the jaguar/obelix mutant., Synopsis Animals display a variety of skin pigment patterns. How these often intricate patterns are formed, however, is the longstanding question. Zebrafish is the only model organism having a pigment pattern, and thus it provides a unique system in which to investigate the mechanism of pattern formation. The striped pigment pattern of zebrafish comprises two types of pigment cells, melanophores (black chromatophores) and xanthophores (yellow chromatophores), and defects in pigment cell differentiation cause abnormal pigment patterns. However, the mechanism(s) underlying the arrangement of pigmented cells during development is unclear. In this paper, the authors cloned and studied the zebrafish mutant gene jaguar/obelix and identified it as inwardly rectifying potassium channel 7.1 (Kir7.1). Although the development of pigment cells is normal in jaguar/obelix fish, they have abnormally wide body stripes; thus, cell positioning is altered, suggesting that the jaguar/obelix functions in the system that determines pigment patterning. The connection between the Kir7.1 channel and the pigment pattern remains unclear, but the mutant melanophores are defective in intracellular aggregation and dispersion of the melanosome (pigment) controlled by the sympathetic neuron, suggesting that the signaling pathway activated by the neuron is also related to pigment pattern formation.

Viruses and transposons are efficient tools for permanently delivering foreign DNA into vertebrate genomes but exhibit diminished activity when cargo exceeds 8 kilobases (kb). This size restriction limits their molecular genetic and biotechnological utility, such as numerous therapeutically relevant genes that exceed 8 kb in size. Furthermore, a greater payload capacity vector would accommodate more sophisticated cis cargo designs to modulate the expression and mutagenic risk of these molecular therapeutics. We show that the Tol2 transposon can efficiently integrate DNA sequences larger than 10 kb into human cells. We characterize minimal sequences necessary for transposition (miniTol2) in vivo in zebrafish and in vitro in human cells. Both the 8.5-kb Tol2 transposon and 5.8-kb miniTol2 engineered elements readily function to revert the deficiency of fumarylacetoacetate hydrolase in an animal model of hereditary tyrosinemia type 1. Together, Tol2 provides a novel nonviral vector for the delivery of large genetic payloads for gene therapy and other transgenic applications., Synopsis Mobile genetic elements (transposons) are effective vehicles for the delivery of foreign DNA for gene therapy and gene discovery applications. Their utility in vertebrates has been, however, limited to relatively few known elements with high activity, including the engineered element Sleeping Beauty (SB) and the naturally occurring fish transposon, Tol2. The authors explore and systematically unlock some of the potential of Tol2, characterizing a minimal set of transposon sequences required for gene transfer by the Tol2-encoding enzyme, transposase. The authors further demonstrate full activity of this “mini” element in human tissue culture cells and in the treatment of a mouse model of tyrosinemia. Tol2 demonstrates high cargo-capacity, readily transferring large (at least 10,000 base pairs) DNA sequences, an ability that opens the door to an array of molecular genetic approaches in vertebrates previously difficult or impossible using prior tools.

Small interfering RNAs (siRNAs), the guides that direct RNA interference (RNAi), provide a powerful tool to reduce the expression of a single gene in human cells. Ideally, dominant, gain-of-function human diseases could be treated using siRNAs that specifically silence the mutant disease allele, while leaving expression of the wild-type allele unperturbed. Previous reports suggest that siRNAs can be designed with single nucleotide specificity, but no rational basis for the design of siRNAs with single nucleotide discrimination has been proposed. We systematically identified siRNAs that discriminate between the wild-type and mutant alleles of two disease genes: the human Cu, Zn superoxide dismutase (SOD1) gene, which contributes to the progression of hereditary amyotrophic lateral sclerosis through the gain of a toxic property, and the huntingtin (HTT) gene, which causes Huntington disease when its CAG-repeat region expands beyond approximately 35 repeats. Using cell-free RNAi reactions in Drosophila embryo lysate and reporter assays and microarray analysis of off-target effects in cultured human cells, we identified positions within an siRNA that are most sensitive to mismatches. We also show that purine:purine mismatches imbue an siRNA with greater discriminatory power than other types of base mismatches. siRNAs in which either a G:U wobble or a mismatch is located in the “seed” sequence, the specialized siRNA guide region responsible for target binding, displayed lower levels of selectivity than those in which the mismatch was located 3′ to the seed; this region of an siRNA is critical for target cleavage but not siRNA binding. Our data suggest that siRNAs can be designed to discriminate between the wild-type and mutant alleles of many genes that differ by just a single nucleotide., Synopsis First discovered in nematodes, RNA interference (RNAi) has become an essential tool in the study of mammalian gene function. RNAi directed by small interfering RNAs (siRNAs), 21 nt, double-stranded RNAs target complementary mRNAs for destruction. siRNAs can be introduced into mammalian cells grown in culture, or even administered intravenously to rodents or primates, where they repress production of the targeted gene product. Thus, siRNA-directed RNAi has tremendous potential as a human therapeutic strategy. Dominant genetic disorders, in which a mutant allele of a gene causes disease in the presence of a second, normal copy, might be treated with therapeutic siRNAs, provided that the siRNAs could be designed to destroy the mutant, disease-causing mRNA, while leaving the normal mRNA intact. Here, Schwarz and colleagues describe an experimentally validated strategy for the design of such siRNAs. Their design strategy should facilitate the design of siRNAs targeting dominant genetic disorders such as amyotrophic lateral sclerosis and Huntington disease.

Piglets appear to lack brown adipose tissue, a specific type of fat that is essential for nonshivering thermogenesis in mammals, and they rely on shivering as the main mechanism for thermoregulation. Here we provide a genetic explanation for the poor thermoregulation in pigs as we demonstrate that the gene for uncoupling protein 1 (UCP1) was disrupted in the pig lineage. UCP1 is exclusively expressed in brown adipose tissue and plays a crucial role for thermogenesis by uncoupling oxidative phosphorylation. We used long-range PCR and genome walking to determine the complete genome sequence of pig UCP1. An alignment with human UCP1 revealed that exons 3 to 5 were eliminated by a deletion in the pig sequence. The presence of this deletion was confirmed in all tested domestic pigs, as well as in European wild boars, Bornean bearded pigs, wart hogs, and red river hogs. Three additional disrupting mutations were detected in the remaining exons. Furthermore, the rate of nonsynonymous substitutions was clearly elevated in the pig sequence compared with the corresponding sequences in humans, cattle, and mice, and we used this increased rate to estimate that UCP1 was disrupted about 20 million years ago., Synopsis Brown adipose tissue (BAT) is unique to mammals. It is rich in mitochondria and generates heat to maintain body temperature during cold stress, referred to as nonshivering thermogenesis. BAT is found in abundance in rodents as well as in newborns of larger mammals, including humans. Uncoupling protein 1 (UCP1) is exclusively expressed in BAT and is localized to the inner membrane of the mitochondria. Its physiological role is to uncouple oxidative phosphorylation so that most of the energy in fat stores is dissipated as heat rather than being converted to ATP. Piglets are sensitive to cold stress and rely on shivering as the main mechanism for thermoregulation. Furthermore, pigs are the only hoofed mammals that build nests for birth; in modern pig production, heat-producing lamps are used to keep the piglets warm. It is also striking that pigs appear to lack BAT. Here the authors show that the UCP1 gene is disrupted in domestic pigs and their wild ancestors. The inactivation of UCP1 was estimated to have happened about 20 million years ago. The finding provides an explanation for the poor thermoregulation in piglets that may have led to the evolution of the unique maternal behavior in this species.

The beginning of this millennium has seen dramatic advances in genomic research. Milestones such as the complete sequencing of the human genome and of many other species were achieved and complemented by the systematic discovery of variation at the single nucleotide (SNP) and whole segment (copy number polymorphism) level. Currently most genomics research efforts are concentrated on the production of whole genome functional annotations, as well as on mapping the epigenome by identifying the methylation status of CpGs, mainly in CpG islands, in different tissues. These recent advances have a major impact on the way genetic research is conducted and have accelerated the discovery of genetic factors contributing to disease. Technology was the critical driving force behind genomics projects: both the combination of Sanger sequencing with high-throughput capillary electrophoresis and the rapid advances in microarray technologies were keys to success. MALDI-TOF MS-based genome analysis represents a relative newcomer in this field. Can it establish itself as a long-term contributor to genetics research, or is it only suitable for niche areas and for laboratories with a passion for mass spectrometry? In this review, we will highlight the potential of MALDI-TOF MS-based tools for resequencing and for epigenetics research applications, as well as for classical complex genetic studies, allele quantification, and quantitative gene expression analysis. We will also identify the current limitations of this approach and attempt to place it in the context of other genome analysis technologies. © 2006 Ragoussis et al.

We present here the results of forward and reverse genetic screens for chemically-induced mutations in Xenopus tropicalis. In our forward genetic screen, we have uncovered 77 candidate phenotypes in diverse organogenesis and differentiation processes. Using a gynogenetic screen design, which minimizes time and husbandry space expenditures, we find that if a phenotype is detected in the gynogenetic F2 of a given F1 female twice, it is highly likely to be a heritable abnormality (29/29 cases). We have also demonstrated the feasibility of reverse genetic approaches for obtaining carriers of mutations in specific genes, and have directly determined an induced mutation rate by sequencing specific exons from a mutagenized population. The Xenopus system, with its well-understood embryology, fate map, and gain-of-function approaches, can now be coupled with efficient loss-of-function genetic strategies for vertebrate functional genomics and developmental genetics., Synopsis Amphibian embryos can be used to understand how all vertebrates, including mammals, develop from fertilized single-celled eggs to establish a body plan and form different cell types and functional organs. Genetic methods are used to analyze what goes wrong in embryos lacking working versions of individual genes, and help to understand those genes' specific functions. However, genetic analysis of previously studied amphibians has been difficult because of these species' long generation time and complex genetic structure. The authors have established methods for systematically studying disrupted genes in the frog Xenopus tropicalis, which has a relatively short generation time, simple genetic structure, and an easily studied externally-developing embryo. They describe their methods for creating and characterizing X. tropicalis mutations, using both forward genetics (where a mutation's effects on the embryo are first characterized, then the DNA defect is later identified) and reverse genetics (where animals carrying mutations in a known DNA sequence are first identified, and the effects of that mutation are characterized subsequently). Studies of amphibian development using tissue culture, transplantation, and molecular tools have been fundamental to understanding vertebrate early development. These studies will be greatly enriched by the addition of forward and reverse genetics to complement emerging genomic tools.

The international FANTOM consortium aims to produce a comprehensive picture of the mammalian transcriptome, based upon an extensive cDNA collection and functional annotation of full-length enriched cDNAs. The previous dataset, FANTOM2, comprised 60,770 full-length enriched cDNAs. Functional annotation revealed that this cDNA dataset contained only about half of the estimated number of mouse protein-coding genes, indicating that a number of cDNAs still remained to be collected and identified. To pursue the complete gene catalog that covers all predicted mouse genes, cloning and sequencing of full-length enriched cDNAs has been continued since FANTOM2. In FANTOM3, 42,031 newly isolated cDNAs were subjected to functional annotation, and the annotation of 4,347 FANTOM2 cDNAs was updated. To accomplish accurate functional annotation, we improved our automated annotation pipeline by introducing new coding sequence prediction programs and developed a Web-based annotation interface for simplifying the annotation procedures to reduce manual annotation errors. Automated coding sequence and function prediction was followed with manual curation and review by expert curators. A total of 102,801 full-length enriched mouse cDNAs were annotated. Out of 102,801 transcripts, 56,722 were functionally annotated as protein coding (including partial or truncated transcripts), providing to our knowledge the greatest current coverage of the mouse proteome by full-length cDNAs. The total number of distinct non-protein-coding transcripts increased to 34,030. The FANTOM3 annotation system, consisting of automated computational prediction, manual curation, and final expert curation, facilitated the comprehensive characterization of the mouse transcriptome, and could be applied to the transcriptomes of other species.

The formation of chimeric gene structures provides important routes by which novel proteins and functions are introduced into genomes. Signatures of these events have been identified in organisms from wide phylogenic distributions. However, the ability to characterize the early phases of these evolutionary processes has been difficult due to the ancient age of the genes or to the limitations of strictly computational approaches. While examples involving retrotransposition exist, our understanding of chimeric genes originating via illegitimate recombination is limited to speculations based on ancient genes or transfection experiments. Here we report a case of a young chimeric gene that has originated by illegitimate recombination in Drosophila. This gene was created within the last 2–3 million years, prior to the speciation of Drosophila simulans, Drosophila sechellia, and Drosophila mauritiana. The duplication, which involved the Bällchen gene on Chromosome 3R, was partial, removing substantial 3′ coding sequence. Subsequent to the duplication onto the X chromosome, intergenic sequence was recruited into the protein-coding region creating a chimeric peptide with ~ 33 new amino acid residues. In addition, a novel intron-containing 5′ UTR and novel 3′ UTR evolved. We further found that this new X-linked gene has evolved testes-specific expression. Following speciation of the D. simulans complex, this novel gene evolved lineage-specifically with evidence for positive selection acting along the D. simulans branch., Synopsis Illegitimate recombination, the non-homologous recombination that occurs between DNA sequences with few or no identical nucleotides, is a general phenomenon that has been known to cause many medically important deleterious changes. However, little is known about the positive side of such a process. For example, little is known about its relative role in the origin of new gene functions that confer increased fitness to species. This work contributes to the understanding of the significance of this process. Here the authors report on a young chimeric gene that has originated by illegitimate recombination in Drosophila. The term “chimeric gene” refers to gene structures—both coding and noncoding—which have been generated from distinct parental loci. This chimeric gene was created within the last 2–3 million years, prior to the speciation of Drosophila simulans, Drosophila sechellia, and Drosophila mauritiana. A gene on Chromosome 3R was duplicated onto the X chromosome and recruited intergenic sequence, creating a chimeric peptide. It was found that this new X-linked gene has evolved testes-specific expression. Following speciation of the D. simulans complex, this novel gene evolved lineage-specifically under positive Darwinian selection.

An important but largely unmet challenge in understanding the mechanisms that govern the formation of specific organs is to decipher the complex and dynamic genetic programs exhibited by the diversity of cell types within the tissue of interest. Here, we use an integrated genetic, genomic, and computational strategy to comprehensively determine the molecular identities of distinct myoblast subpopulations within the Drosophila embryonic mesoderm at the time that cell fates are initially specified. A compendium of gene expression profiles was generated for primary mesodermal cells purified by flow cytometry from appropriately staged wild-type embryos and from 12 genotypes in which myogenesis was selectively and predictably perturbed. A statistical meta-analysis of these pooled datasets—based on expected trends in gene expression and on the relative contribution of each genotype to the detection of known muscle genes—provisionally assigned hundreds of differentially expressed genes to particular myoblast subtypes. Whole embryo in situ hybridizations were then used to validate the majority of these predictions, thereby enabling true-positive detection rates to be estimated for the microarray data. This combined analysis reveals that myoblasts exhibit much greater gene expression heterogeneity and overall complexity than was previously appreciated. Moreover, it implicates the involvement of large numbers of uncharacterized, differentially expressed genes in myogenic specification and subsequent morphogenesis. These findings also underscore a requirement for considerable regulatory specificity for generating diverse myoblast identities. Finally, to illustrate how the developmental functions of newly identified myoblast genes can be efficiently surveyed, a rapid RNA interference assay that can be scored in living embryos was developed and applied to selected genes. This integrated strategy for examining embryonic gene expression and function provides a substantially expanded framework for further studies of this model developmental system., Synopsis Animal development requires cells in complex organs to acquire distinct identities. During the development of the body wall musculature of the fruit fly, a pool of apparently identical cells gives rise to two types of muscle precursors, both of which are required for the appearance of functioning muscles. These identities depend on broad programs of gene expression. The authors attempt to dissect the complements of expressed genes that define these two different cell types by integrating modern methods in genetics, genomics, and informatics. By purifying informative cells from normal embryos and mutants that perturb muscle development, assaying their genomewide gene expression programs, and combining experiments statistically, they have identified fivefold more founder-specific genes than were previously suspected to characterize this cell type. The expression patterns of hundreds of genes were examined in whole embryos to test the statistical predictions, permitting the authors to estimate how many more cell type–specific genes remain to be discovered. Finally, dozens of the genes highlighted by these methods were tested for direct involvement in muscle development, and several new players in this process are reported. The integrated strategy used here can be generalized for studying genetic programs in other complex tissues.

Acinetobacter baumannii is a species of nonfermentative gram-negative bacteria commonly found in water and soil. This organism was susceptible to most antibiotics in the 1970s. It has now become a major cause of hospital-acquired infections worldwide due to its remarkable propensity to rapidly acquire resistance determinants to a wide range of antibacterial agents. Here we use a comparative genomic approach to identify the complete repertoire of resistance genes exhibited by the multidrug-resistant A. baumannii strain AYE, which is epidemic in France, as well as to investigate the mechanisms of their acquisition by comparison with the fully susceptible A. baumannii strain SDF, which is associated with human body lice. The assembly of the whole shotgun genome sequences of the strains AYE and SDF gave an estimated size of 3.9 and 3.2 Mb, respectively. A. baumannii strain AYE exhibits an 86-kb genomic region termed a resistance island—the largest identified to date—in which 45 resistance genes are clustered. At the homologous location, the SDF strain exhibits a 20 kb-genomic island flanked by transposases but devoid of resistance markers. Such a switching genomic structure might be a hotspot that could explain the rapid acquisition of resistance markers under antimicrobial pressure. Sequence similarity and phylogenetic analyses confirm that most of the resistance genes found in the A. baumannii strain AYE have been recently acquired from bacteria of the genera Pseudomonas, Salmonella, or Escherichia. This study also resulted in the discovery of 19 new putative resistance genes. Whole-genome sequencing appears to be a fast and efficient approach to the exhaustive identification of resistance genes in epidemic infectious agents of clinical significance., Synopsis The bacterial species Acinetobacter baumannii is a major cause of hospital-acquired infection throughout the world, and it is an increasing public health concern due to its increasing resistance to antibiotic treatment. Coincidently, a high incidence of multidrug-resistant A. baumannii bloodstream infections was recently reported in US Army service members injured during Afghanistan and Iraq/Kuwait military operations. A. baumannii exhibits a remarkable ability to rapidly develop antibiotic resistance, which led from fully susceptible to multidrug-resistant strains within three decades. The authors used whole-genome sequencing and bioinformatic analyses to identify the complete repertoire of resistance genes exhibited by the multidrug-resistant A. baumannii strain AYE, which is epidemic in France, and to investigate the mechanisms of their acquisition by comparison with the fully susceptible A. baumannii strain SDF, which is associated with human body lice. This study led to the discovery in the AYE genome of an 86-kb region called a resistance “island”—the largest identified to date—that contains a cluster of 45 resistance genes. The homologous location in the susceptible strain, curiously, exhibited a 20-kb genomic island that is devoid of resistance markers. This ability to “switch” its genomic structure probably explains the unmatched speed at which A. baumannii captures resistance markers when under antibacterial pressure, such as is found in hospital intensive care units.

High phenotypic variation in diet-induced obesity in male C57BL/6J inbred mice suggests a molecular model to investigate non-genetic mechanisms of obesity. Feeding mice a high-fat diet beginning at 8 wk of age resulted in a 4-fold difference in adiposity. The phenotypes of mice characteristic of high or low gainers were evident by 6 wk of age, when mice were still on a low-fat diet; they were amplified after being switched to the high-fat diet and persisted even after the obesogenic protocol was interrupted with a calorically restricted, low-fat chow diet. Accordingly, susceptibility to diet-induced obesity in genetically identical mice is a stable phenotype that can be detected in mice shortly after weaning. Chronologically, differences in adiposity preceded those of feeding efficiency and food intake, suggesting that observed difference in leptin secretion is a factor in determining phenotypes related to food intake. Gene expression analyses of adipose tissue and hypothalamus from mice with low and high weight gain, by microarray and qRT-PCR, showed major changes in the expression of genes of Wnt signaling and tissue re-modeling in adipose tissue. In particular, elevated expression of SFRP5, an inhibitor of Wnt signaling, the imprinted gene MEST and BMP3 may be causally linked to fat mass expansion, since differences in gene expression observed in biopsies of epididymal fat at 7 wk of age (before the high-fat diet) correlated with adiposity after 8 wk on a high-fat diet. We propose that C57BL/6J mice have the phenotypic characteristics suitable for a model to investigate epigenetic mechanisms within adipose tissue that underlie diet-induced obesity., Synopsis Genetic models to explain the obesity epidemic are inadequate because the emergence of this epidemic over the past 30 y has been too rapid to allow for the appearance of new mutant genes. The authors show that diet-induced obesity among genetically identical mice is characterized by highly variable and stable phenotypes that are established in mice early in life, even before they become exposed to an obesogenic environment. Furthermore, strong associations occur between susceptibility to obesity and the expression of genes implicated in processes that regulate cellular development. Previous studies have shown that abnormal regulation of such genes by epigenetic mechanisms is linked with the development of cancer. Epigenetic mechanisms involve chemical processes that change chromatin structure and gene expression without changing the genetic code. Accordingly, epigenetic modifications of gene structure through nutritional and physiological stress provide mechanisms for inducing obesity that are independent of new mutations to the genome. Experimental models based upon genetically identical mice provide powerful tools for identifying epigenetic and environmental mechanisms causing obesity and other chronic diseases.

Many proteins are used repeatedly in development, but usually the function of the protein is similar in the different contexts. Here we report that the classical Drosophila melanogaster locus tan encodes a novel enzyme required for two very different cellular functions: hydrolysis of N-β-alanyl dopamine (NBAD) to dopamine during cuticular melanization, and hydrolysis of carcinine to histamine in the metabolism of photoreceptor neurotransmitter. We characterized two tan-like P-element insertions that failed to complement classical tan mutations. Both are inserted in the 5′ untranslated region of the previously uncharacterized gene CG12120, a putative homolog of fungal isopenicillin-N N-acyltransferase (EC 2.3.1.164). Both P insertions showed abnormally low transcription of the CG12120 mRNA. Ectopic CG12120 expression rescued tan mutant pigmentation phenotypes and caused the production of striking black melanin patterns. Electroretinogram and head histamine assays indicated that CG12120 is required for hydrolysis of carcinine to histamine, which is required for histaminergic neurotransmission. Recombinant CG12120 protein efficiently hydrolyzed both NBAD to dopamine and carcinine to histamine. We conclude that D. melanogaster CG12120 corresponds to tan. This is, to our knowledge, the first molecular genetic characterization of NBAD hydrolase and carcinine hydrolase activity in any organism and is central to the understanding of pigmentation and photoreceptor function., Synopsis True et al. describe the identification and characterization of the Drosophila melanogaster enzyme Tan. The gene encoding Tan was originally discovered in the early 20th century as a mutant strain lacking the dark pigmentation of wild-type flies, hence the name tan. Flies lacking Tan function also exhibited mysterious abnormalities in vision, for example, in responses to light. The new findings by True et al. help to explain the vastly different functions of Tan in pigmentation and vision. In the developing epidermal cells that secrete the adult cuticle, the enzyme encoded by tan is required for the production of dopamine, which is needed for dark melanin pigmentation. In the eye, the Tan enzyme converts carcinine, a modified form of the neurotransmitter histamine, back to histamine, which is necessary for the rapid and constant neurotransmission events involved in vision. These two enzyme activities have not been previously characterized in any organism. Surprisingly, Tan appears to be closely related to an enzyme in fungi that is used for production of the antibiotic penicillin.

Artemisinins, derived from the wormwood herb Artemisia annua, are the most potent antimalarial drugs currently available. Despite extensive research, the exact mode of action of artemisinins has not been established. Here we use yeast, Saccharamyces cerevisiae, to probe the core working mechanism of this class of antimalarial agents. We demonstrate that artemisinin's inhibitory effect is mediated by disrupting the normal function of mitochondria through depolarizing their membrane potential. Moreover, in a genetic study, we identify the electron transport chain as an important player in artemisinin's action: Deletion of NDE1 or NDI1, which encode mitochondrial NADH dehydrogenases, confers resistance to artemisinin, whereas overexpression of NDE1 or NDI1 dramatically increases sensitivity to artemisinin. Mutations or environmental conditions that affect electron transport also alter host's sensitivity to artemisinin. Sensitivity is partially restored when the Plasmodium falciparum NDI1 ortholog is expressed in yeast ndi1 strain. Finally, we showed that artemisinin's inhibitory effect is mediated by reactive oxygen species. Our results demonstrate that artemisinin's effect is primarily mediated through disruption of membrane potential by its interaction with the electron transport chain, resulting in dysfunctional mitochondria. We propose a dual role of mitochondria played during the action of artemisinin: the electron transport chain stimulates artemisinin's effect, most likely by activating it, and the mitochondria are subsequently damaged by the locally generated free radicals., Synopsis Malaria kills at least 1 million people worldwide a year. Recent years saw the rapid emergence of drug-resistant malaria strains. Artemisinins, derived from the Chinese wormwood herb Artemisia annua, are the most potent antimalarials currently available. Despite extensive research, the exact mode of action of artemisinins has not been established. In this article, Li et al. investigated yeast as a model to probe the core working mechanism of this class of antimalarials. They showed that artemisinin can disrupt the normal function of mitochondria by depolarizing its membrane potential, and that artemisinin's effect can be affected by its interaction with the mitochondrial electron transport chain, an apparatus that couples oxygen oxidation and energy generation in the cell. They proposed a dual role of mitochondria played during the action of artemisinin: the electron transport chain likely activates artemisinin, and the mitochondria are subsequently damaged by the locally generated free radicals associated with this activation. The research has provided a fine tool for the study of the mechanism of artemisinin in a model organism (yeast), and laid the framework for a set of possible future experiments to be conducted in yeast and malaria parasites.

Argonaute proteins are essential components of the molecular machinery that drives RNA silencing. In Drosophila, different members of the Argonaute family of proteins have been assigned to distinct RNA silencing pathways. While Ago1 is required for microRNA function, Ago2 is a crucial component of the RNA-induced silencing complex in siRNA-triggered RNA interference. Drosophila Ago2 contains an unusual amino-terminus with two types of imperfect glutamine-rich repeats (GRRs) of unknown function. Here we show that the GRRs of Ago2 are essential for the normal function of the protein. Alleles with reduced numbers of GRRs cause specific disruptions in two morphogenetic processes associated with the midblastula transition: membrane growth and microtubule-based organelle transport. These defects do not appear to result from disruption of siRNA-dependent processes but rather suggest an interference of the mutant Ago2 proteins in an Ago1-dependent pathway. Using loss-of-function alleles, we further demonstrate that Ago1 and Ago2 act in a partially redundant manner to control the expression of the segment-polarity gene wingless in the early embryo. Our findings argue against a strict separation of Ago1 and Ago2 functions and suggest that these proteins act in concert to control key steps of the midblastula transition and of segmental patterning., Synopsis Cells employ diverse mechanisms to control the activity of their genes, and over the last ten years, a new strategy for gene regulation called RNA silencing has been discovered. Central components responsible for RNA silencing are Argonaute proteins. While many of the molecular properties of Argonaute proteins have been uncovered, little is known about their function in living organisms. In Drosophila, it has been puzzling that mutations in individual Argonaute proteins lead to surprisingly mild defects in development, although RNA silencing had been suggested to play major roles in gene regulation. Meyer and coworkers describe that Ago1 and Ago2, two Argonaute family members in Drosophila, function in a redundant fashion. Previously, these two proteins were demonstrated to mediate distinct pathways of RNA silencing. The authors show that in early embryos Ago1 and Ago2 work together in two fundamental processes: the generation of polarity within cells, by controlling the unequal distributions of proteins and cell organelles, and the polarity of tissues, by modulating an important cell-cell signaling pathway. These results connect the activity of Argonaute proteins and by extension the mechanisms of RNA silencing with central problems of cell and developmental biology, namely the regulation of polarity in cells and tissues.

Systems biology approaches that are based on the genetics of gene expression have been fruitful in identifying genetic regulatory loci related to complex traits. We use microarray and genetic marker data from an F2 mouse intercross to examine the large-scale organization of the gene co-expression network in liver, and annotate several gene modules in terms of 22 physiological traits. We identify chromosomal loci (referred to as module quantitative trait loci, mQTL) that perturb the modules and describe a novel approach that integrates network properties with genetic marker information to model gene/trait relationships. Specifically, using the mQTL and the intramodular connectivity of a body weight–related module, we describe which factors determine the relationship between gene expression profiles and weight. Our approach results in the identification of genetic targets that influence gene modules (pathways) that are related to the clinical phenotypes of interest., Synopsis Obesity is a major pub lic health concern in many developed countries. While some people appear to stay lean no matter what or how much they eat, others appear to be genetically predisposed to obesity. The genetic similarity between mouse and human makes the mouse a promising mammalian model system to study obesity. Advantages of mouse models include the ability to control diet/environment and easy access to relevant tissues for gene expression studies. Mouse cross studies have implicated dozens of chromosomal regions that contain weight-predisposing genes, and gene expression studies have yielded hundreds of body weight–related genes. In this study, the authors use a gene network–based approach for integrating clinical traits, genetic marker data, and gene expression data. Instead of focusing on individual genes, the authors provide a systems-level view of a module of genes related to body weight. The resulting model allows them to characterize weight-related genes utilizing network concepts (intramodular connectivity) and genetic concepts (module quantitative trait locus). This integrative genomics approach provides new insights into the relationship between gene expression and body weight.

The major histocompatibility complex (MHC) is recognised as one of the most important genetic regions in relation to common human disease. Advancement in identification of MHC genes that confer susceptibility to disease requires greater knowledge of sequence variation across the complex. Highly duplicated and polymorphic regions of the human genome such as the MHC are, however, somewhat refractory to some whole-genome analysis methods. To address this issue, we are employing a bacterial artificial chromosome (BAC) cloning strategy to sequence entire MHC haplotypes from consanguineous cell lines as part of the MHC Haplotype Project. Here we present 4.25 Mb of the human haplotype QBL (HLA-A26-B18-Cw5-DR3-DQ2) and compare it with the MHC reference haplotype and with a second haplotype, COX (HLA-A1-B8-Cw7-DR3-DQ2), that shares the same HLA-DRB1, -DQA1, and -DQB1 alleles. We have defined the complete gene, splice variant, and sequence variation contents of all three haplotypes, comprising over 259 annotated loci and over 20,000 single nucleotide polymorphisms (SNPs). Certain coding sequences vary significantly between different haplotypes, making them candidates for functional and disease-association studies. Analysis of the two DR3 haplotypes allowed delineation of the shared sequence between two HLA class II–related haplotypes differing in disease associations and the identification of at least one of the sites that mediated the original recombination event. The levels of variation across the MHC were similar to those seen for other HLA-disparate haplotypes, except for a 158-kb segment that contained the HLA-DRB1, -DQA1, and -DQB1 genes and showed very limited polymorphism compatible with identity-by-descent and relatively recent common ancestry (, Synopsis A group of genes involved in the human immune system are contained within a surprisingly short section of Chromosome 6 that has long been recognised as the most important genomic region in relation to disease susceptibility. Discerning the actual genes playing a role in disease has proved difficult mainly because the region contains numerous genes and is also the most genetically variable in the genome. Within this jungle of variation, the research reported here has identified and characterised a discrete segment shared by two individuals that is virtually devoid of variation—a polymorphism desert. The conservation of this segment amongst a background of extreme variation suggests both an ancient origin and genetic exchange in early human history. These observations are important in evolutionary terms as they reveal a potential mechanism whereby certain genetic segments associated with favourable immune functions have spread across human populations. Within medical terms this may also explain contrasting disease risks in people from different ethnic backgrounds. Public access to these data will help researchers find specific variants conferring disease susceptibility or resistance and, as in this report, rule out regions for conveying specificity to certain diseases.

Quantitative traits are conditioned by several genetic determinants. Since such genes influence many important complex traits in various organisms, the identification of quantitative trait loci (QTLs) is of major interest, but still encounters serious difficulties. We detected four linked genes within one QTL, which participate in controlling sporulation efficiency in Saccharomyces cerevisiae. Following the identification of single nucleotide polymorphisms by comparing the sequences of 145 genes between the parental strains SK1 and S288c, we analyzed the segregating progeny of the cross between them. Through reciprocal hemizygosity analysis, four genes, RAS2, PMS1, SWS2, and FKH2, located in a region of 60 kilobases on Chromosome 14, were found to be associated with sporulation efficiency. Three of the four “high” sporulation alleles are derived from the “low” sporulating strain. Two of these sporulation-related genes were verified through allele replacements. For RAS2, the causative variation was suggested to be a single nucleotide difference in the upstream region of the gene. This quantitative trait nucleotide accounts for sporulation variability among a set of ten closely related winery yeast strains. Our results provide a detailed view of genetic complexity in one “QTL region” that controls a quantitative trait and reports a single nucleotide polymorphism-trait association in wild strains. Moreover, these findings have implications on QTL identification in higher eukaryotes., Synopsis Genes controlling many medically and agriculturally important complex traits in various organisms and their organization as quantitative trait loci (QTLs) are of major interest. To identify QTLs responsible for such a quantitative trait, the authors employed a two-step strategy: First, single-nucleotide markers (called SNPs) distributed throughout the genome were screened for prevalence among progeny with extreme characteristics, thus identifying three candidate genomic regions. Next, in one of these regions, manipulation of individual genes revealed four tightly linked genes that affected the trait, sporulation efficiency. A fifth gene that affects sporulation was recently and independently identified in the same region. This 60-kilobase region has a complex and interesting architecture: One strain, which sporulates efficiently, has sporulation-promoting alleles (alternative forms) at two major genes and inhibiting alleles at the three less important ones, whereas another strain, with inefficient sporulation, has the opposite alleles at the five genes. Moreover, one causative SNP for this trait, in the promoter region of the gene RAS2, explains sporulation differences among a set of ten winery yeast strains. These results provide a detailed view of genetic complexity in one “QTL region” and an SNP-trait association example among wild strains.

Nonsyndromic or isolated cleft lip with or without cleft palate (CL/P) occurs in wide geographic distribution with an average birth prevalence of 1/700. We used direct sequencing as an approach to study candidate genes for CL/P. We report here the results of sequencing on 20 candidate genes for clefts in 184 cases with CL/P selected with an emphasis on severity and positive family history. Genes were selected based on expression patterns, animal models, and/or role in known human clefting syndromes. For seven genes with identified coding mutations that are potentially etiologic, we performed linkage disequilibrium studies as well in 501 family triads (affected child/mother/father). The recently reported MSX1 P147Q mutation was also studied in an additional 1,098 cleft cases. Selected missense mutations were screened in 1,064 controls from unrelated individuals on the Centre d'Étude du Polymorphisme Humain (CEPH) diversity cell line panel. Our aggregate data suggest that point mutations in these candidate genes are likely to contribute to 6% of isolated clefts, particularly those with more severe phenotypes (bilateral cleft of the lip with cleft palate). Additional cases, possibly due to microdeletions or isodisomy, were also detected and may contribute to clefts as well. Sequence analysis alone suggests that point mutations in FOXE1, GLI2, JAG2, LHX8, MSX1, MSX2, SATB2, SKI, SPRY2, and TBX10 may be rare causes of isolated cleft lip with or without cleft palate, and the linkage disequilibrium data support a larger, as yet unspecified, role for variants in or near MSX2, JAG2, and SKI. This study also illustrates the need to test large numbers of controls to distinguish rare polymorphic variants and prioritize functional studies for rare point mutations., Synopsis Nonsyndromic or isolated cleft lip with or without cleft palate (CL/P) is a birth defect with wide geographic distribution, occurring with an average frequency of 1/700 live births. Treatment can be provided, but it will involve medical, surgical, dental, and psychological personnel. Several different genes have been implicated in different cases. Here the researchers report the results of sequencing 20 different genes in 184 CL/P cases selected with an emphasis on more severe cases and cases with a positive family history for CL/P. Genes were selected based on previous work done by others and by the researchers' group. The authors' results suggest that point mutations in these candidate genes are likely to contribute to about 5% of CL/P, and particularly those with more severe phenotypes (bilateral cleft of the lip with cleft palate). This study also illustrates the need to test large numbers of controls to distinguish rare polymorphic variants and allow investigators to focus functional studies on the rare point mutations that seem to be disease-causing, so that researchers might better understand the mechanisms that play a role in CL/P.

Congenital nephrogenic diabetes insipidus (NDI) is a disease characterized by failure of the kidney to concentrate urine in response to vasopressin. Human kindreds with nephrogenic diabetes insipidus have been found to harbor mutations in the vasopressin receptor 2 (Avpr2) gene or the vasopressin-sensitive water channel aquaporin-2 (Aqp2) gene. Development of a treatment is rendered difficult due to the lack of a viable animal model. Through forward genetic screening of ethylnitrosourea-mutagenized mice, we report the identification and characterization of a mouse model of NDI, with an F204V mutation in the Aqp2 gene. Unlike previously attempted murine models of NDI, our mice survive to adulthood and more exactly recapitulate the human disorder. Previous in vitro experiments using renal cell lines suggest recessive Aqp2 mutations result in improper trafficking of the mutant water pore. Using these animals, we have directly proven this hypothesis of improper AQP2 translocation as the molecular defect in nephrogenic diabetes insipidus in the intact organism. Additionally, using a renal cell line we show that the mutated protein, AQP2-F204V, is retained in the endoplasmic reticulum and that this abnormal localization can be rescued by wild-type protein. This novel mouse model allows for further mechanistic studies as well as testing of pharmacological and gene therapies for NDI., Synopsis Nephrogenic diabetes insipidus (NDI) is a disease marked by excessive urination and thirst. Normally, the hypothalamus senses situations where water is limited and signals to the kidney to increase water reabsorption from urine. The signaling molecule secreted by the hypothalamus is arginine vasopressin (AVP), which binds to a specific protein on the surface of kidney cells, AVP receptor (AVPR2). AVP binding to its receptor on kidney cells begins a series of biochemical events that ultimately results in the insertion of a protein, aquaporin 2 (AQP2), into the outer surface of the kidney cell. As its name suggests, AQP2 facilitates the reuptake of water from the urinary space into the cell, thus concentrating the urine and conserving water. Congenital NDI is caused by mutations in either the water channel, AQP2, or in the receptor, AVPR2. While these mutations have been studied extensively in the lab, work in live animals has been very limited. This report describes the first viable mouse model of NDI. Previous models have been attempted by targeted mutation, i.e., genes known to be involved in the disease have been altered in the mouse, a so-called reverse genetic approach. Reverse genetic approaches have so far failed to produce a viable mouse model of NDI. Here the authors take a forward genetic approach in which genes are mutated at random and animals are screened for disease-like properties. As well as proving hypotheses that come from lab studies, this model opens the door to the testing of gene therapy or other therapies for treatment of NDI.

Despite its importance in harboring genes critical for spermatogenesis and male-specific functions, the Y chromosome has been largely excluded as a priority in recent mammalian genome sequencing projects. Only the human and chimpanzee Y chromosomes have been well characterized at the sequence level. This is primarily due to the presumed low overall gene content and highly repetitive nature of the Y chromosome and the ensuing difficulties using a shotgun sequence approach for assembly. Here we used direct cDNA selection to isolate and evaluate the extent of novel Y chromosome gene acquisition in the genome of the domestic cat, a species from a different mammalian superorder than human, chimpanzee, and mouse (currently being sequenced). We discovered four novel Y chromosome genes that do not have functional copies in the finished human male-specific region of the Y or on other mammalian Y chromosomes explored thus far. Two genes are derived from putative autosomal progenitors, and the other two have X chromosome homologs from different evolutionary strata. All four genes were shown to be multicopy and expressed predominantly or exclusively in testes, suggesting that their duplication and specialization for testis function were selected for because they enhance spermatogenesis. Two of these genes have testis-expressed, Y-borne copies in the dog genome as well. The absence of the four newly described genes on other characterized mammalian Y chromosomes demonstrates the gene novelty on this chromosome between mammalian orders, suggesting it harbors many lineage-specific genes that may go undetected by traditional comparative genomic approaches. Specific plans to identify the male-specific genes encoded in the Y chromosome of mammals should be a priority., Synopsis Y chromosomes are typically gene poor and enriched with repetitive elements, making them difficult to sequence by standard methods. Hence, the Y chromosome gene repertoire in mammalian species other than human has not been explored until very recently. Here the authors used a directed approach to isolate Y chromosome genes of the domestic cat, an evolutionary divergent species from human and mouse. They found that the feline Y chromosome harbors its own unique set of genes that are expressed specifically in the testes, presumably where they play an important role in spermatogenesis. Paralleling the discoveries seen from the full human Y chromosome sequence, the feline Y chromosome has acquired and remodeled some genes from autosomes, while other genes have a shared ancestry with the X chromosome. However, none of the four new genes are found on the Y chromosomes of human or mouse, although two are shared with the canine Y chromosome. This work highlights the Y chromosome as a source of potential gene novelty in different species and suggests that more directed efforts at characterizing this hitherto understudied chromosome will further enrich our understanding of the types of genes found there and the roles they may play in mammalian spermatogenesis.

Size control is essential for all proliferating cells, and is thought to be regulated by checkpoints that couple cell size to cell cycle progression. The aberrant cell-size phenotypes caused by mutations in the retinoblastoma (RB) tumor suppressor pathway are consistent with a role in size checkpoint control, but indirect effects on size caused by altered cell cycle kinetics are difficult to rule out. The multiple fission cell cycle of the unicellular alga Chlamydomonas reinhardtii uncouples growth from division, allowing direct assessment of the relationship between size phenotypes and checkpoint function. Mutations in the C. reinhardtii RB homolog encoded by MAT3 cause supernumerous cell divisions and small cells, suggesting a role for MAT3 in size control. We identified suppressors of an mat3 null allele that had recessive mutations in DP1 or dominant mutations in E2F1, loci encoding homologs of a heterodimeric transcription factor that is targeted by RB-related proteins. Significantly, we determined that the dp1 and e2f1 phenotypes were caused by defects in size checkpoint control and were not due to a lengthened cell cycle. Despite their cell division defects, mat3, dp1, and e2f1 mutants showed almost no changes in periodic transcription of genes induced during S phase and mitosis, many of which are conserved targets of the RB pathway. Conversely, we found that regulation of cell size was unaffected when S phase and mitotic transcription were inhibited. Our data provide direct evidence that the RB pathway mediates cell size checkpoint control and suggest that such control is not directly coupled to the magnitude of periodic cell cycle transcription., Synopsis All cell types have a characteristic size, but the means by which cell size is determined remain mysterious. In proliferating cells, control mechanisms termed checkpoints are thought to prevent cells from dividing until they have reached a minimum size, but the nature of size checkpoints has proved difficult to dissect. The unicellular alga Chlamydomonas reinhardtii divides via an unusual mechanism that uncouples growth from division, and thereby allows a direct assessment of how different genetic pathways contribute to size control. The retinoblastoma (RB) tumor suppressor pathway is a critical regulator of cell cycle control in plants and animals and is thought to act as a transcriptional switch for cell cycle genes, but it had not been directly implicated in cell size checkpoint function. The authors found that mutations in genes that encode key proteins of the RB pathway in Chlamydomonas affect cell size and cell cycle control by altering size checkpoint function. Unexpectedly, the predicted transcriptional targets of the RB pathway were not affected by the mutations, and blocking transcription did not alter cell size control. These data link the RB tumor suppressor pathway directly to size control and suggest the possibility that cell size and cell cycle control by the RB pathway may not be coupled to its transcriptional output.