Search

Suberin has been shown to play a role in protection against and response to stress, including defense against soil-borne pathogens. In soybean, oxidized fatty acids are the most predominant monomers contributing to root suberin, with 18-hydroxy-oleic acid being the most abundant. 18-Hydroxy-oleic acid is predicted to be synthesized by members of the CYP86A family of cytochrome P450 enzyme. Six putative CYP86A genes were identified through phylogenetic analysis of the soybean genome. Two of these, CYP86A37 and CYP86A38 show a root-specific gene expression pattern, and were further analyzed to assess their physiological role in suberin deposition using RNAi knockdown in a hairy root system. Soybean hairy roots were found to be nearly identical to soil-grown roots in terms of anatomy, suberin deposition patterns, and suberin chemistry. The RNAi knockdown of CYP86A37 and CYP86A38 yielded hairy root lines with reduced gene expression and a reduction in the oxidized monomers of suberin, most notably 18-hydroxy-oleic acid. Based on this evidence, CYP86A37 and CYP86A38 are predicted to function as fatty acid [omega]-hydroxylases in vivo. Keywords: Glycine max, soybean, hairy root, suberin, cytochrome P450, CYP86A37, CYP86A38. La subérine s'avère jouer un rôle dans la protection et la réponse au stress, notamment dans la défense contre des agents pathogènes du sol. Chez le soja, les acides gras oxydés sont les principaux monomères qui contribuent à la subérine racinaire, l'acide 18-hydroxy-oléique étant le plus abondant. On prédit que l'acide 18-hydroxyoléique sera synthétisé par les membres de la famille CYP86Adu cytochrome P450. Six gènes présumés du CYP86A ont été identifiés au moyen de l'analyse phylogénétique du génome du soja. Deux d'entre eux, CYP86A37 et CYP86A38 présentent un profil d'expression génique spécifique aux racines et ont été analysés de manière plus approfondie afin d'évaluer leur rôle physiologique dans le dépôt de la subérine, à l'aide d'un knockdown par ARNi dans un système de culture aérienne de racines. Les racines de soja en culture aérienne se sont avérées presque identiques aux racines cultivées en terre en matière d'anatomie, de profils de dépôt de subérine et de chimie de la subérine. Le knockdown des CYP86A37 et CYP86A38 par ARNi produisait des lignées de racines aériennes avec une expression génique réduite et une diminution des monomères oxydés de subérine, plus particulièrement de l'acide 18-hydroxy-oléique. À partir de ces données, on prévoit que les CYP86A37 et CYP86A38 agissent comme [omega]-hydroxylases d'acides gras in vivo. [Traduit par la Rédaction] Mots-clés : Glycine max, soja, racine en culture aérienne, subérine, cytochrome P450, CYP86A37, CYP86A38., Introduction Soybean [Glycine max (L.) Merr.] is an oilseed crop widely cultivated for food, feed, and renewable energy production. However, like all crop plants, soybean production is impacted by both [...]

We show that phage lysogenization, lysogens, and prophage induction are all targeted by CRISPR. The results demonstrate that genomic DNA is not immune to the CRISPR system, that the CRISPR system does not require noncytoplasmic elements, and that the system protects from phages entering and exiting the lysogenic cycle. doi: 10.1128/JB.00644-10

The soil actinomycete Kocuria rhizophila belongs to the suborder Micrococcineae, a divergent bacterial group for which only a limited amount of genomic information is currently available. K. rhizophila is also important in industrial applications; e.g., it is commonly used as a standard quality control strain for antimicrobial susceptibility testing. Sequencing and annotation of the genome of K. rhizophila DC2201 (NBRC 103217) revealed a single circular chromosome (2,697,540 bp; G+C content of 71.16%) containing 2,357 predicted protein-coding genes. Most of the predicted proteins (87.7%) were orthologous to actinobacterial proteins, and the genome showed fairly good conservation of synteny with taxonomically related actinobacterial genomes. On the other hand, the genome seems to encode much smaller numbers of proteins necessary for secondary metabolism (one each of nonribosomal peptide synthetase and type III polyketide synthase), transcriptional regulation, and lateral gene transfer, reflecting the small genome size. The presence of probable metabolic pathways for the transformation of phenolic compounds generated from the decomposition of plant materials, and the presence of a large number of genes associated with membrane transport, particularly amino acid transporters and drug efflux pumps, may contribute to the organism's utilization of root exudates, as well as the tolerance to various organic compounds.

Abstract: Recent genome size estimates for Arctic amphipods have revealed the largest genomes known in the Crustacea. Here we provide additional data for 7 species of caridean shrimp collected from [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.030 Byline: Manisha Goel (a), Arcady Mushegian (a)(b) Keywords: S. purpuratus; Sea urchin; Comparative genomics; Metabolic pathways; Thiaminase; Urea cycle; Cholesterol; Phosphoglyerate mutase Abstract: The genome sequence of the purple sea urchin Strongylocentrotus purpuratus recently became available. We report the results of functional annotation and initial analysis of more than 2300 proteins predicted to be involved in metabolite transport and enzymatic conversion in sea urchin. The comparison of various reconstructed biosynthetic and catabolic pathways in sea urchin to those known in other genomes suggests the overall similarity of the sea urchin metabolism to that of the vertebrates, with relatively small but non-trivial differences from both vertebrates and protostomes. There are several examples of two parallel, non-orthologous solutions for the same molecular function in sea urchin, in contrast with the other completely sequenced metazoans that tend to contain just one version of the same function. There are also genes that appear to be close phylogenetic neighbors of plant or bacterial homologs, as opposed to homologs in other Metazoa. The evolutionary and functional significance of these variations is discussed. Author Affiliation: (a) Stowers Institute for Medical Research, 1000 E. 50th St., Kansas City, MO 64110, USA (b) Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City, KS 66160, USA Article History: Received 8 May 2006; Revised 10 August 2006; Accepted 16 August 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.09.012 Byline: Antonio Fernandez-Guerra (a), Antoine Aze (a), Julia Morales (b), Odile Mulner-Lorillon (b), Bertrand Cosson (b), Patrick Cormier (b), Cynthia Bradham (c), Nikki Adams (d), Anthony J. Robertson (e), William F. Marzluff (f), James A. Coffman (e), Anne-Marie Geneviere (a) Keywords: Sea urchin; Cell cycle; Replication; Mitosis; Checkpoint; Kinases Abstract: A search of the Strongylocentrotus purpuratus genome for genes associated with cell cycle control and DNA metabolism shows that the known repertoire of these genes is conserved in the sea urchin, although with fewer family members represented than in vertebrates, and with some cases of echinoderm-specific gene diversifications. For example, while homologues of the known cyclins are mostly encoded by single genes in S. purpuratus (unlike vertebrates, which have multiple isoforms), there are additional genes encoding novel cyclins of the B and K/L types. Almost all known cyclin-dependent kinases (CDKs) or CDK-like proteins have an orthologue in S. purpuratus; CDK3 is one exception, whereas CDK4 and 6 are represented by a single homologue, referred to as CDK4. While the complexity of the two families of mitotic kinases, Polo and Aurora, is close to that found in the nematode, the diversity of the NIMA-related kinases (NEK proteins) approaches that of vertebrates. Among the nine NEK proteins found in S. purpuratus, eight could be assigned orthologues in vertebrates, whereas the ninth is unique to sea urchins. Most known DNA replication, DNA repair and mitotic checkpoint genes are also present, as are homologues of the pRB (two) and p53 (one) tumor suppressors. Interestingly, the p21/p27 family of CDK inhibitors is represented by one homologue, whereas the INK4 and ARF families of tumor suppressors appear to be absent, suggesting that these evolved only in vertebrates. Our results suggest that, while the cell cycle control mechanisms known from other animals are generally conserved in sea urchin, parts of the machinery have diversified within the echinoderm lineage. The set of genes uncovered in this analysis of the S. purpuratus genome should enhance future research on cell cycle control and developmental regulation in this model. Author Affiliation: (a) Observatoire Oceanologique de Banyuls-Laboratoire Arago, CNRS-UMR7628/UPMC, 66650 Banyuls-sur-Mer, France (b) Station Biologique de Roscoff, CNRS-UMR7150/UPMC, 29680 Roscoff, France (c) Developmental, Cellular, and Molecular Biology Group, Duke University, Durham, NC, USA (d) Department of Biological Sciences, California Polytechnic State University, San Luis Obispo, CA, USA (e) Mount Desert Island Biological Laboratory, Salisbury Cove, ME, USA (f) Program in Molecular Biology, University of North Carolina, Chapel Hill, NC, USA Article History: Received 30 May 2006; Revised 6 September 2006; Accepted 7 September 2006

The main virulence factor of Vibrio cholerae, the cholera toxin, is encoded by the ctxAB operon, which is contained in the genome of the lysogenic filamentous phage CTX[phi]. This phage transmits ctxAB genes between V. cholerae bacterial populations that express toxin-coregulated pilus (TCP), the CTX[phi] receptor. In investigating new forms of ctxAB transmission, we found that V. cholerae filamentous phage VGJ[phi], which uses the mannose-sensitive hemagglutinin (MSHA) pilus as a receptor, transmits CTX[phi] or its satellite phage RS1 by an efficient and highly specific TCP-independent mechanism. This is a novel type of specialized transduction consisting in the site-specific cointegration of VGJ[phi] and CTX[phi] (or RS1) replicative forms to produce a single hybrid molecule, which generates a single-stranded DNA hybrid genome that is packaged into hybrid viral particles designated HybP[phi] (for the VGJ[phi]/CTX[phi] hybrid) and HybRS[phi] (for the VGJ[phi]/RS1 hybrid). The hybrid phages replicate by using the VGJ[phi] replicating functions and use the VGJ[phi] capsid, retaining the ability to infect via MSHA. The hybrid phages infect most tested strains more efficiently than CTX[phi], even under in vitro optimal conditions for TCP expression. Infection and lysogenization with HybP[phi] revert the V. cholerae live attenuated vaccine strain 1333 to virulence. Our results reinforce that TCP is not indispensable for the acquisition of CTX[phi]. Thus, we discuss an alternative to the current accepted evolutionary model for the emergence of new toxigenic strains of V. cholerae and the importance of our findings for the development of an environmentally safer live attenuated cholera vaccine.

Several streptococcal species are able to take up naked DNA from the environment and integrate it into their genomes by homologous recombination. This process is called natural transformation. In Streptococcus pneumoniae and related streptococcal species, competence for natural transformation is induced by a peptide pheromone through a quorum-sensing mechanism. Recently we showed that induction of the competent state initiates lysis and release of DNA from a subfraction of the bacterial population and that the efficiency of this process is influenced by cell density. Here we have further investigated the nature of this cell density-dependent release mechanism. Interestingly, we found that competence-induced pneumococci lysed competence-deficient cells of the same strain during cocultivation and that the efficiency of this heterolysis increased as the ratio of competent to noncompetent cells increased. Furthermore, our results indicate that the lysins made by competent pneumococci are not released into the growth medium. More likely, they are anchored to the surface of the competent cells by choline-binding domains and cause lysis of noncompetent pneumococci through cell-to-cell contact.

Pathogenicity of the gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria depends on a type III secretion (TTS) system which translocates bacterial elector proteins into the plant cell. Previous transcriptome analysis identified a genome-wide regulon of putative virulence genes that are coexpressed with the TTS system. In this study, we characterized two of these genes, xopC and xopJ. Both genes encode Xanthomonas outer proteins (Xops) that were shown to be secreted by the TTS system. In addition, type III-dependent translocation of both proteins into the plant cell was demonstrated using the AvrBs3 effector domain as a reporter. XopJ belongs to the AvrRxv/YopJ family of effector proteins from plant and animal pathogenic bacteria. By contrast, XopC does not share significant homology to proteins in the database. Sequence analysis revealed that the xopC locus contains several features that are reminiscent of pathogenicity islands. Interestingly, the xopC region is flanked by 62-bp inverted repeats that are also associated with members of the Xanthomonas avrBs3 effector family. Besides xopC, a second gene of the locus, designated hpaJ, was shown to be coexpressed with the TTS system. hpaJ encodes a protein with similarity to transglycosylases and to the Pseudomonas syringae pv. maculicola protein HopPmaG. HpaJ secretion and translocation by the X. campestris pv. vesicatoria TTS system was not detectable, which is consistent with its predicted Sec signal and a putative function as transglycosylase in the bacterial periplasm.

We report here the construction of a physical and genetic map of the virulent Wolbachia strain, wMelPop. This map was determined by ordering 28 chromosome fragments that resulted from digestion with the restriction endonucleases FseI, ApaI, SmaI, and AscI and were resolved by pulsed-field gel electrophoresis. Southern hybridization was done with 53 Wolbachia-specific genes as probes in order to determine the relative positions of these restriction fragments and use them to serve as markers. Comparison of the resulting map with the whole genome sequence of the closely related benign Wolbachia strain, wMel, shows that the two genomes are largely conserved in gene organization with the exception of a single inversion in the chromosome.

The bacterial genome projects have suggested a central role for horizontal transfer in bacterial adaptation, but it is difficult to rule out an adaptive role for ordinary genetic change in existing genes. The bacterial systematics literature can readily address the importance of gene acquisition in adaptive evolution, since phenotypic characterization typically assesses presence versus absence of metabolic capabilities, and metabolic gains and losses are most likely due to horizontal transfer and/or gene loss. Bacterial systematists have not geared their studies toward quantitative differences in metabolic capabilities, which are more likely to involve adjustments of existing genes. Here, quantitative variation in metabolism within and between three closely related Bacillus taxa has been assayed. While these taxa show no qualitative (i.e. presence versus absence) differences in resource utilization, they are quantitatively different in utilization of 8% of 95 resources tested. Moreover, 93% of the resources tested showed significant quantitative variation among strains within a single taxon. These results suggest that ordinary genetic changes in existing genes may play an important role in adaptation. If these results are typical, future genomically based assays of quantitative variation in phenotype (e.g. microarray analysis of mRNA concentrations) may identify hundreds of genes whose expression has been modified. A protocol is presented for identifying those modifications of gene expression and those gene acquisitions that are most likely to have played a role in adaptive evolution.

The availability of an ever increasing number of complete genome sequences of diverse prokaryotic taxa has led to the introduction of novel approaches to infer phylogenetic relationships among bacteria. In the present study the sequences of the 16S rRNA gene and nine housekeeping genes were compared with the fraction of shared putative orthologous protein-encoding genes, conservation of gene order, dinucteotide relative abundance and codon usage among 11 genomes of species belonging to the lactic acid bacteria. In general there is a good correlation between the results obtained with various approaches, although it is clear that there is a stronger phylogenetic signal in some datasets than in others, and that different parameters have different taxonomic resolutions. It appears that trees based on different kinds of information derived from whole-genome sequencing projects do not provide much additional information about the phylogenetic relationships among bacterial taxa compared to more traditional alignment-based methods. Nevertheless, it is expected that the study of these novel forms of information will have its value in taxonomy, to determine which genes are shared, when genes or sets of genes were lost in evolutionary history, to detect the presence of horizontally transferred genes and/or confirm or enhance the phylogenetic signal derived from traditional methods. Although these conclusions are based on a relatively small dataset, they are largely in agreement with other studies and it is anticipated that similar trends will be observed when comparing other genomes.

The direct binding of Streptococcus mitis to human platelets is mediated in part by two proteins (PblA and PblB) encoded by a lysogenic bacteriophage (SM1). Since SM1 is the first prophage of S. mitis that has been identified and because of the possible role of these phage-encoded proteins in virulence, we sought to characterize SMI in greater detail. Sequencing of the SM1 genome revealed that it consisted of 34,692 bp, with an overall G+C content of 39 mol%. Fifty-six genes encoding proteins of 40 or more amino acids were identified. The genes of SMI appear to be arranged in a modular, life cycle-specific organization. BLAST analysis also revealed that the proteins of SM1 have homologies to proteins from a wide variety of lambdoid phages. Bioinformatic analyses, in addition to N-terminal sequencing of the proteins, led to the assignment of possible functions to a number of proteins, including the integrase, the terminase, and two major structural proteins. Examination of the phage structural components indicates that the phage head may assemble using stable multimers of the major capsid protein, in a process similar to that of phage r1t. These findings indicate that SMI may be part of a discrete subfamily of the Siphoviridae that includes at least phages r1t of Lactococcus lactis and SF370.3 of Streptococcus pyogenes.

Mycobacterium ulcerans causes Buruli ulcer, the third most prevalent mycobacterial infection of immunocompetent humans after tuberculosis and leprosy. Recent work has shown that the production by M. ulcerans of mycolactone, a novel polyketide, may partly explain the pathogenesis of Buruli ulcer. To search for the genetic basis of virulence in M. ulcerans, we took advantage of the close genetic relationship between M. ulcerans and Mycobacterium marinum by performing genomic suppressive subtractive hybridization of M. ulcerans with M. marinum. We identified several DNA fragments specific to M. ulcerans, in particular, a type I polyketide synthase locus with a highly repetitive modular arrangement. We postulate that this locus is responsible for the synthesis of mycolactone in M. ulcerans.

The nitrifying bacterium Nitrosomonas europaea can obtain all its carbon for growth from C[O.sub.2] and all its energy and reductant for growth from the oxidation of N[H.sub.3] and is considered an obligate chemolithoautotroph. Previous studies have shown that N. europaea can utilize limited amounts of certain organic compounds, including amino acids, pyruvate, and acetate, although no organic compound has been reported to support the growth of N. europaea. The recently completed genomic sequence of N. europaea revealed a potential permease for fructose. With this in mind, we tested if N. europaea could utilize fructose and other compounds as carbon sources to support growth. Cultures were incubated in the presence of fructose or other organic compounds in sealed bottles purged of C[O.sub.2]. In these cultures, addition of either fructose or pyruvate as the sole carbon source resulted in a two-to threefold increase in optical density and protein content in 3 to 4 days. Studies with [[sub.14]C] fructose showed that >90% of the carbon incorporated by the cells during growth was derived from fructose. Cultures containing mannose, glucose, glycerol, mannitoi, citrate, or acetate showed little or no growth. N. europaea was not able to grow with fructose as an energy source, although the presence of fructose did provide an energy benefit to the cells. These results show that N. europaea can be grown in C[O.sub.2]-free medium by using fructose and pyruvate as carbon sources and may now be considered a facultative chemolithoorganotroph.

Ureases are multisubunit enzymes requiring [Ni.sup.2+] for activity. The low pH-inducible urease gene cluster in Streptococcus salivarius 57.I is organized as an operon, beginning with ureI, followed by ureABC (structural genes), and ureEFGD (accessory genes). Urease biogenesis also requires a high-affinity [Ni.sup.2+] uptake system. By searching the partial genome sequence of a closely related organism, Streptococcus thermophilus LMG18311, three open reading frame (ORFs) homologous to those encoding proteins involved in cobalamin biosynthesis and cobalt transport (cbiMQO) were identified immediately 3' to the ure operon. To determine whether these genes were involved in urease biogenesis by catalyzing [Ni.sup.2+] uptake in S. salivarius, regions 3' to ureD were amplified by PCRs from S. salivarius by using primers identical to the S. thermophilus sequences. Sequence analysis of the products revealed three ORFs. Reverse transcriptase PCR was used to demonstrate that the ORFs are transcribed as part of the ure operon. Insertional inactivation of ORF1 with a polar kanamycin marker completely abolished urease activity and the ability to accumulate [.sup.63][Ni.sup.2+] during growth. Supplementation of the growth medium with Ni[Cl.sup.2] at concentrations as low as 2.5 [micro]M partially restored urease activity in the mutant. Both wild-type and mutant strains showed enhanced urease activity when exogenous [Ni.sup.2+] was provided at neutral pH. Enhancement of urease activity by adding nickel was regulated at the posttranslational level. Thus, ORF1, ORF2, and ORF3 are part of the ure operon, and these genes, designated ureM, ureQ, and ureO, respectively, likely encode a [Ni.sup.2+]-specific ATP-binding cassette transporter.

Increasing knowledge of the genome sequences of several organisms and the development of genome-wide, high-throughput screening techniques for gene expression are likely to generate a vast amount of data aimed at elucidating the molecular mechanisms of addiction. These findings are likely to have potential for future addiction pharmacotherapies. However, it is important to employ animal models that dissociate the molecular and cellular consequences of the direct pharmacological effects of addictive drugs from those that result from the cognitive processes associated with self-administration of these drugs. In this article, we suggest that the short-term and long-term neuroadaptive effects of addictive drugs in the brain depend crucially on the drug-exposure paradigm used [i.e. passive (non-contingent) drug exposure and active (contingent) self-administration]. This has important ramifications for future molecular and cellular studies of drug addiction.

Over 150 mutations with documented pathogenicity have been identified within the human mitochondrial genome. More than half of the disease-related mutations are located within tRNA genes, a remarkable trend, given that these sequences comprise only 10% of the genome. The discovery of diseases correlated with mitochondrial tRNA mutations provides the first example of a class of pathologies related to RNA function, and the study of these tRNAs provides an interesting opportunity to explore the relationship between physiology and tRNA function. Investigations of both cellular and molecular effects have provided important insights into the structural and functional defects caused by the mutations. The picture that emerges from varied studies is that the effects of tRNA mutations are probably multifaceted and complex, but can be traced to the destabilization of structural features that destroy the native tRNA fold required for all aspects of function.

This study investigates the prevalence of CG and CNG suppression in single- vs. multicopy DNA regions of the maize genome. The analysis includes the single- and multicopy seed storage proteins (zeins), the miniature inverted-repeat transposable elements (MITEs), and long terminal repeat (LTR) retrotransposons. Zein genes are clustered on specific chromosomal regions, whereas MITEs and LTRs are dispersed in the genome. The multicopy zein genes are CG suppressed and exhibit large variations in CG suppression. The variation observed correlates with the extent of duplication each zein gene has undergone, indicating that gene duplication results in an increased turnover of cytosine residues. Alignment of individual zein genes confirms this observation and demonstrates that CG depletion results primarily from polarized C:T and G:A transition mutations from a less to a more extensively duplicated gene. In addition, transition mutations occur primarily in a CG or CNG context suggesting that CG suppression may result from deamination of methylated cytosine residues. Duplication-dependent CG depletion is likely to occur at other loci as duplicated MITEs and LTR elements, or elements inserted into duplicated gene regions, also exhibit CG depletion.

An RFLP genomic subtraction was used to isolate male-specific sequences in the species Silene latifolia. One isolated fragment, SLP2, shares similarity to a portion of the Activator (Ac) transposase from Zea mays and to related proteins from other plant species. Southern blot analysis of male and female S. latifolia genomic DNA shows that SLP2 belongs to a low-copy-number repeat family with two Y-linked copies. Screening of a S. latifolia male genomic library using SLP2 as a probe led to the isolation of five clones, which were partially sequenced. One clone contains two large open reading frames that can be joined into a sequence encoding a putative protein of 682 amino acids by removing a short intron. Database searches and phylogenetic analysis show that this protein belongs to the hAT superfamily of transposases, closest to Tag2 (Arabidopsis thaliana), and contains all of the defined domains critical for the activity of these transposases. PCR with genomic and cDNA templates from S. latifolia male, female, and hermaphrodite individuals revealed that one of the Y-linked copies is transcriptionally active and alternatively spliced. This is the first report of a transcriptionally active transposable element (TE) family in S. latifolia and the first DNA transposon residing on a plant Y chromosome. The potential activity and regulation of this TE family and its use for Y chromosome gene discovery is discussed.

Fluorescent in situ hybridization was used to examine the distribution of six abundant long terminal repeat (LTR) retroelements, Opie, Huck, Cinful-1, Prem-2/Ji, Grande, and Tekay/Prem-1 on maize pachytene chromosomes. Retroelement staining in euchromatin was remarkably uniform, even when we included the structurally polymorphic abnormal chromosome 10 (Abl0) in our analysis. This uniformity made it possible to use euchromatin as a control for quantitative staining intensity measurements in other regions of the genome. The data show that knobs, known to function as facultative neocentromeres when Abl0 is present, tend to exclude retroelements. A notable exception is Cinful-1, which accumulates in TR-1 knob arrays. Staining for each of the six retroelements was also substantially reduced in centromeric satellite arrays to an average of 30% of the staining in euchromatin. This contrasted with two previously described centromere-specific retrotransposable (CR) elements that were readily detected in centromeres. We suggest that retroelements are relatively rare in centromeres because they interrupt the long satellite arrays thought to be required for efficient centromere function. CR elements may have evolved mutualistic relationships with their plant hosts: they are known to interact with the kinetochore protein CENH3 and appear to accumulate in clusters, leaving long satellite arrays intact.

Within the unique Triplo-lethal region (Tpl) of the Drosophila melanogaster genome we have found a cluster of 20 genes encoding a novel family of proteins. This family is also present in the Anopheles gambiae genome and displays remarkable synteny and sequence conservation with the Drosophila cluster. The family is also present in the sequenced genome of D. pseudoobscura, and homologs have been found in Aedes aegypti mosquitoes and in four other insect orders, but it is not present in the sequenced genome of any noninsect species. Phylogenetic analysis suggests that the cluster evolved prior to the divergence of Drosophila and Anopheles (250 MYA) and has been highly conserved since. The ratio of synonymous to nonsynonymous substitutions and the high codon bias suggest that there has been selection on this family both for expression level and function. We hypothesize that this gene family is Tpl, name it the Osiris family, and consider possible functions. We also predict that this family of proteins, due to the unique dosage sensitivity and the lack of homologs in noninsect species, would be a good target for genetic engineering or novel insecticides.

Cdh1p, a substrate specificity factor for the cell cycle-regulated ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), promotes exit from mitosis by directing the degradation of a number of proteins, including the mitotic cyclins. Here we present evidence that Cdh1p activity at the M/[G.sub.1] transition is important not only for mitotic exit but also for high-fidelity chromosome segregation in the subsequent cell cycle. CDH1 showed genetic interactions with MAD2 and PDS1, genes encoding components of the mitotic spindle assembly checkpoint that acts at metaphase to prevent premature chromosome segregation. Unlike cdh1[DELTA] and mad2[DELTA] single mutants, the mad2[DELTA] cdh1[DELTA] double mutant grew slowly and exhibited high rates of chromosome and plasmid loss. Simultaneous deletion of PDS1 and EDH1 caused extensive chromosome missegregation and cell death. Our data suggest that at least part of the chromosome loss can be attributed to kinetochore/spindle problems. Our data further suggest that Cdh1p and Sic1p, a Cdc28p/Clb inhibitor, have overlapping as well as nonoverlapping roles in ensuring proper chromosome segregation. The severe growth defects of both mad2[DELTA] cdh1[DELTA] and pds1[DELTA] cdh1[DELTA] strains were rescued by overexpressing Swe1p, a [G.sub.2]/M inhibitor of the cyclin-dependent kinase, Cdc28p/Clb. We propose that the failure to degrade cyclins at the end of mitosis leaves cdh1[DELTA] mutant strains with abnormal Cdc28p/ Clb activity that interferes with proper chromosome segregation.

Diverse types of genomic DNA organization have been found in Rhizobiaceae, especially among Agrobacterium species. Previous studies of Agrobactereum concentrated mainly on biovar 1 strains. Little attention has been given to biovar 2 strains. The biovar 2 genome consists of a large, circular chromosome and second megabase-sized replicon, as well as several plasmids. In this study two biovar 2 strains were analysed, A. rhizogenes (A. radiobacter) K84 and A. rhizogenes A4, by constructing physical maps of their chromosomes and mega-replicons. The maps revealed that in both strains their chromosomes consist of approximately 3.7 Mbp, while the mega-replicons are 2.6 Mbp circular DNAs. Gene mapping and comparative genomic analysis were performed based on the physical maps using Southern hybridization. It was found that rDNA, as well as analysed virulence and virulence-related genes, are present only on the chromosomes. The inter-chromosomal relationship between biovar 1 and biovar 2 strains was also analysed. Interestingly, there was a high similarity between the chromosomes of biovar 2 and the circular chromosomes of biovar 1, whereas similarity among the smaller megabase-sized replicons was restricted to each biovar. Based on these observations the possible relationship among large replicons in Agrobacterium biovars 1 and 2 is discussed.

Temporal transcription of phage [phi]adh was analysed during lyric reproduction. Based on Northern hybridizations the phage genome was divided into regions ct early, middle and late transcription. Eight groups of overlapping transcripts, probably originating from common precursors, were distinguished. Early transcription of a 10.9 kb region adjacent to the lytic/ lysogenic switch started within the first 10 rain of infection and produced three groups of mRNAs mostly related to DNA replication. Four middle transcripts were observed 30 min after infection, corresponding to an 8.5 kb genomic region, which started at the replication origin (ori) and encompassed a DNA packaging function and the cos site. Three groups of late transcripts were first observed 50 min after infection, corresponding to a 21.1 kb region between the middle region and the attachment site (attP), encoding functions for capsid morphogenesis and host cell lysis. A fourth group of late-appearing mRNAs was divergently transcribed from the 3.2 kb section between attP and the lytic/lysogenic switch, including the repressor and integrase genes. Except for one set of early mRNAs, all the transcripts persisted until the end of the reproduction cycle. Two confirmed and two predicted promoters were assigned to transcript 5' ends in the early region.

Members of the Mycobacterium avium complex are distinguished by the presence of highly antigenic surface molecules called glycopeptidolipids (GPLs) and the oligosaccharide portion of the serovar-specific GPL defines the 28 serovars. Previously, the genomic region (ser2) encoding the enzymes responsible for the glycosylation of the lipopeptide core to generate the serovar-2-specific GPLs has been described. In this work, the ser2 gene clusters of M. avium serovar 2 strains 2151 and TMC 724 were fully sequenced and compared to the homologous regions of M. avium serovar 1 strain 104, M. avium subsp, paratuberculosis and M. avium subsp. silvaticum. It was also determined that 104Rg, a mutant of strain 104 that produces truncated GPLs, lost several GPL biosynthesis genes by deletion. This comparison, together with analysis of protein similarities, supports a biosynthetic model in which serovar-2-specific GPLs are synthesized from a serovar-1-specific GPL intermediate that is derived from a non-specific GPL precursor. We also identified a gene encoding an enzyme that is necessary for the biosynthesis of serovar-3-and 9-specific GPLs, but not serovar-2-specific GPLs, suggesting that the different serovars may have evolved from the acquisition or loss of genetic information, in addition, a subcluster of genes for the biosynthesis and transfer of fucose, which are needed to make serovar-specific GPLs such as those of serovar 2, is found in the non-GPL-producing M. avium subspecies paratuberculosis and silvaticum.

Staphylococcus aureus binds to human desquamated nasal epithelial cells, a phenomenon likely to be important in nasal colonization. ClfB was identified previously as one staphylococcal adhesin that promoted binding to nasal epithelia. In this study, it is shown that the S. aureus surface protein SasG, identified previously by in silico analysis of genome sequences, and two homologous proteins, Pls of S. aureus and AAP of Staphylococcus epidermidis, also promote bacterial adherence to nasal epithelial cells. Conditions for in vitro expression of SasG by S. aureus were not found. Adherence assays were therefore performed with S. aureus and Lactococcus lactis expressing SasG from an expression plasmid. These studies showed that SasG did not bind several ligands typically bound by S. aureus. Significantly, SasG and Pls did promote bacterial adherence to nasal epithelial cells. Furthermore, pre-incubation of epithelial ceils with purified recombinant proteins revealed that the N-terminal A regions of SasG, Pls and AAP, but not the B repeats of SasG, inhibited adherence of L. lactis expressing SasG in a dose-dependent fashion. These results suggest that SasG, Pls and AAP bind to the same as-yet-unidentified receptor on the surface of nasal epithelial cells, In addition, expression of SasG, like Pls, reduced adherence of S. aureus to fibronectin and fibrinogen.