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Basal metabolic rate generally scales with body mass in mammals, and variation from predicted levels indicates adaptive metabolic remodeling. As a thermogenic adaptation for living in cool water, sea otters have a basal metabolic rate approximately three times that of the predicted rate; however, the tissue-level source of this hypermetabolism is unknown. Because skeletal muscle is a major determinant of whole-body metabolism, we characterized respiratory capacity and thermogenic leak in sea otter muscle. Compared with that of previously sampled mammals, thermogenic muscle leak capacity was elevated and could account for sea otter hypermetabolism. Muscle respiratory capacity was modestly elevated and reached adult levels in neonates. Premature metabolic development and high leak rate indicate that sea otter muscle metabolism is regulated by thermogenic demand and is the source of basal hypermetabolism., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.

Genetics aims to understand the relation between genotype and phenotype. However, because complete deletion of most yeast genes ( approximately 80%) has no obvious phenotypic consequence in rich medium, it is difficult to study their functions. To uncover phenotypes for this nonessential fraction of the genome, we performed 1144 chemical genomic assays on the yeast whole-genome heterozygous and homozygous deletion collections and quantified the growth fitness of each deletion strain in the presence of chemical or environmental stress conditions. We found that 97% of gene deletions exhibited a measurable growth phenotype, suggesting that nearly all genes are essential for optimal growth in at least one condition.

Cryptococcus neoformans is a basidiomycetous yeast ubiquitous in the environment, a model for fungal pathogenesis, and an opportunistic human pathogen of global importance. We have sequenced its approximately 20-megabase genome, which contains approximately 6500 intron-rich gene structures and encodes a transcriptome abundant in alternatively spliced and antisense messages. The genome is rich in transposons, many of which cluster at candidate centromeric regions. The presence of these transposons may drive karyotype instability and phenotypic variation. C. neoformans encodes unique genes that may contribute to its unusual virulence properties, and comparison of two phenotypically distinct strains reveals variation in gene content in addition to sequence polymorphisms between the genomes.

Functional analysis of a genome requires accurate gene structure information and a complete gene inventory. A dual experimental strategy was used to verify and correct the initial genome sequence annotation of the reference plant Arabidopsis. Sequencing full-length cDNAs and hybridizations using RNA populations from various tissues to a set of high-density oligonucleotide arrays spanning the entire genome allowed the accurate annotation of thousands of gene structures. We identified 5817 novel transcription units, including a substantial amount of antisense gene transcription, and 40 genes within the genetically defined centromeres. This approach resulted in completion of approximately 30% of the Arabidopsis ORFeome as a resource for global functional experimentation of the plant proteome.

Molecular genetic studies of Drosophila melanogaster have led to profound advances in understanding the regulation of development. Here we report gene expression patterns for nearly one-third of all Drosophila genes during a complete time course of development. Mutations that eliminate eye or germline tissue were used to further analyze tissue-specific gene expression programs. These studies define major characteristics of the transcriptional programs that underlie the life cycle, compare development in males and females, and show that large-scale gene expression data collected from whole animals can be used to identify genes expressed in particular tissues and organs or genes involved in specific biological and biochemical processes.

Oligonucleotide microarrays were used to map the detailed topography of chromosome replication in the budding yeast Saccharomyces cerevisiae. The times of replication of thousands of sites across the genome were determined by hybridizing replicated and unreplicated DNAs, isolated at different times in S phase, to the microarrays. Origin activations take place continuously throughout S phase but with most firings near mid-S phase. Rates of replication fork movement vary greatly from region to region in the genome. The two ends of each of the 16 chromosomes are highly correlated in their times of replication. This microarray approach is readily applicable to other organisms, including humans.

The scarcity of usable nitrogen frequently limits plant growth. A tight metabolic association with rhizobial bacteria allows legumes to obtain nitrogen compounds by bacterial reduction of dinitrogen (N2) to ammonium (NH4+). We present here the annotated DNA sequence of the alpha-proteobacterium Sinorhizobium meliloti, the symbiont of alfalfa. The tripartite 6.7-megabase (Mb) genome comprises a 3.65-Mb chromosome, and 1.35-Mb pSymA and 1.68-Mb pSymB megaplasmids. Genome sequence analysis indicates that all three elements contribute, in varying degrees, to symbiosis and reveals how this genome may have emerged during evolution. The genome sequence will be useful in understanding the dynamics of interkingdom associations and of life in soil environments.

Locomotor activity by diving marine mammals is accomplished while breath-holding and often exceeds predicted aerobic capacities. Video sequences of freely diving seals and whales wearing submersible cameras reveal a behavioral strategy that improves energetic efficiency in these animals. Prolonged gliding (greater than 78% descent duration) occurred during dives exceeding 80 meters in depth. Gliding was attributed to buoyancy changes with lung compression at depth. By modifying locomotor patterns to take advantage of these physical changes, Weddell seals realized a 9.2 to 59.6% reduction in diving energetic costs. This energy-conserving strategy allows marine mammals to increase aerobic dive duration and achieve remarkable depths despite limited oxygen availability when submerged.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

The hunting behavior of a marine mammal was studied beneath the Antarctic fast ice with an animal-borne video system and data recorder. Weddell seals stalked large Antarctic cod and the smaller subice fish Pagothenia borchgrevinki, often with the under-ice surface for backlighting, which implies that vision is important for hunting. They approached to within centimeters of cod without startling the fish. Seals flushed P. borchgrevinki by blowing air into subice crevices or pursued them into the platelet ice. These observations highlight the broad range of insights that are possible with simultaneous recordings of video, audio, three-dimensional dive paths, and locomotor effort.

Analysis of the 1,042,519-base pair Chlamydia trachomatis genome revealed unexpected features related to the complex biology of chlamydiae. Although chlamydiae lack many biosynthetic capabilities, they retain functions for performing key steps and interconversions of metabolites obtained from their mammalian host cells. Numerous potential virulence-associated proteins also were characterized. Several eukaryotic chromatin-associated domain proteins were identified, suggesting a eukaryotic-like mechanism for chlamydial nucleoid condensation and decondensation. The phylogenetic mosaic of chlamydial genes, including a large number of genes with phylogenetic origins from eukaryotes, implies a complex evolution for adaptation to obligate intracellular parasitism.

As more genomes are sequenced, the identification and characterization of the causes of heritable variation within a species will be increasingly important. It is demonstrated that allelic variation in any two isolates of a species can be scanned, mapped, and scored directly and efficiently without allele-specific polymerase chain reaction, without creating new strains or constructs, and without knowing the specific nature of the variation. A total of 3714 biallelic markers, spaced about every 3.5 kilobases, were identified by analyzing the patterns obtained when total genomic DNA from two different strains of yeast was hybridized to high-density oligonucleotide arrays. The markers were then used to simultaneously map a multidrug-resistance locus and four other loci with high resolution (11 to 64 kilobases).

The genome of the yeast Saccharomyces cerevisiae has been completely sequenced through a worldwide collaboration. The sequence of 12,068 kilobases defines 5885 potential protein-encoding genes, approximately 140 genes specifying ribosomal RNA, 40 genes for small nuclear RNA molecules, and 275 transfer RNA genes. In addition, the complete sequence provides information about the higher order organization of yeast's 16 chromosomes and allows some insight into their evolutionary history. The genome shows a considerable amount of apparent genetic redundancy, and one of the major problems to be tackled during the next stage of the yeast genome project is to elucidate the biological functions of all of these genes.

A high-capacity system was developed to monitor the expression of many genes in parallel. Microarrays prepared by high-speed robotic printing of complementary DNAs on glass were used for quantitative expression measurements of the corresponding genes. Because of the small format and high density of the arrays, hybridization volumes of 2 microliters could be used that enabled detection of rare transcripts in probe mixtures derived from 2 micrograms of total cellular messenger RNA. Differential expression measurements of 45 Arabidopsis genes were made by means of simultaneous, two-color fluorescence hybridization.

The Arabidopsis mutant ttg lacks both trichomes (epidermal hairs) and anthocyanin pigments. Trichomes and anthocyanins are restored by the constitutive expression of the maize transcriptional regulator (R). The expression of an R-glucocorticoid receptor chimeric protein results in a steroid hormone-dependent, conditional allele of R that functions in whole Arabidopsis plants. The response of the chimeric protein to pulses of hormone was used to define the pattern and timing of trichome formation on the developing leaf epidermis. Each adaxial epidermal leaf cell appears to have an equal probability of differentiating into a trichome; there is a temporal zone of decision for trichome cell fate that proceeds as a wave from the tip to the base of developing leaves.

Two Arabidopsis thaliana genes, QRT1 and QRT2, are required for pollen separation during normal development. In qrt mutants, the outer walls of the four meiotic products of the pollen mother cell are fused, and pollen grains are released in tetrads. Pollen is viable and fertile, and the cytoplasmic pollen contents are discrete. Pollination with a single tetrad usually yields four seeds, and genetic analysis confirmed that marker loci segregate in a 2:2 ratio within these tetrads. These mutations allow tetrad analysis to be performed in Arabidopsis and define steps in pollen cell wall development.

Anthocyanin pathway-specific transcriptional activators R and C1 from the monocot maize were expressed in two dicots, Arabidopsis thaliana and Nicotiana tabacum. Expression of R caused augmented anthocyanin pigmentation in both plant species and augmented trichome (hair) production in Arabidopsis. Alone, C1 had no effect. Hybrid transgenic Arabidopsis expressing both C1 and R produced anthocyanins in root, petal, and stamen tissues that normally never express anthrocyanins. When R was expressed in the transparent testa glabrous (without anthocyanins and trichomes) mutant of Arabidopsis, the deficiency was complemented and both anthocyanins and trichomes were restored.

Cold-sensitive mutations in the SPB genes (spb1-spb7) of Saccharomyces cerevisiae suppress the inhibition of translation initiation resulting from deletion of the poly(A)-binding protein gene (PAB1). The SPB4 protein belongs to a family of adenosine triphosphate (ATP)-dependent RNA helicases. The aberrant production of 25S ribosomal RNA (rRNA) occurring in spb4-1 mutants or the deletion of SPB2 (RPL46) permits the deletion of PAB1. These data suggest that mutations affecting different steps of 60S subunit formation can allow PAB-independent translation, and they indicate that further characterization of the spb mutations could lend insight into the biogenesis of the ribosome.

Yeast transformation can be used to insert new sequence arrangements into a variety of chromosomal locations by homologous recombination. These newly inserted sequences can recombine with similar sequences located on other chromosomes. In these events, information is duplicated without being lost at the site from which it is derived. Similar mechanisms might be utilized by cells to provide new functions during development or differentiation.

Unequal crossing-over within a head-to-tail tandem array of the homologous red and green visual pigment genes has been proposed to explain the observed variation in green-pigment gene number among individuals and the prevalence of red-green fusion genes among color-blind subjects. This model was tested by probing the structure of the red and green pigment loci with long-range physical mapping techniques. The loci were found to constitute a gene array with an approximately 39-kilobase repeat length. The position of the red pigment gene at the 5' edge of the array explains its lack of variation in copy number. Restriction maps of the array in four individuals who differ in gene number are consistent with a head-to-tail configuration of the genes. These results provide physical evidence in support of the model and help to explain the high incidence of color blindness in the human population.

The effects of Eco RI endonuclease-cleaved Escherichia coli and yeast (Saccharomyces cerevisiae) DNA fragments on the propagation of the lambda bacteriophage vectors containing them were determined on a nonmutanted and a PolA E. coli K12 host. Observable alterations in the growth of hybrids containing yeast DNA insertions were less frequent and less extreme than those seen in hybrids containing E. coli DNA. A lambda-E. coli hybrid was selected after extensive growth on the Pol A (deficient in polymerase I) host which also grew very well on the PolA+ host and may have resulted from some alteration in the hybrid. Hybrids selected on the PolA host gave no evidence for the expression of polymerase I activity. No lambda-yeast hybrid made from the lambdagt vector lacking lambda-specific recombination (red-) had a yield of viable bacteriophage on infection greater than two-thirds that of "wild-type" lambda.

A lambda vector that contains the gene for Escherichia coli DNA ligase (lambdagt4-lop-11 lig+) has been modified to achieve overproduction of this enzyme. The third Eco RI site in the lambda chromosome has been altered by mutation, and the left-hand Eco RI fragment has been shortened. The new vector, lambdagt4-lop-11 lig+, forms a stable lysogen which, upon induction, produces a 100-fold increase in DNA ligase activity. Introduction of a phage mutation (S7) that prevents cell lysis results in an even greater increase (500-fold).

Electric fields can be manipulated by a method in which multiple electrodes are arranged along a closed contour and clamped to predetermined electric potentials. This method may be applied to a broad range of problems in the separation of macromolecules by gel electrophoresis. DNA molecules as large as 2 megabases can be well separated with a contour-clamped homogeneous electric field alternating between two orientations 120 degrees apart. The pattern of separation is independent of position in the gel, which is an advantage over previous methods. DNA less than 50 kilobases can be separated without distortion even at high voltage with a nonalternating contour-clamped homogeneous field. Decreased band broadening in DNA less than 200 bases can be achieved with a contour-clamped inhomogeneous field.

Genes encoding yeast RNA polymerase II subunits were cloned. Efficient isolation of these genes was accomplished by probing a phage lambda gt11 recombinant DNA expression library with polyvalent antibodies directed against purified yeast RNA polymerase II. The identity of genes that specify the largest RNA polymerase II subunits, the 220,000- and 150,000-dalton polypeptides, was confirmed by competitive radioimmune assay. Both of these genes exist in single copy in the yeast Saccharomyces cerevisiae.

A rapid, direct method for screening single plaques of Agt recombinant phage is described. The method allows at least 10(6) clones to be screened per day and simplifies physical containment of recombinants.

During 4- to 8-day periods at sea, half of 2595 dives of three king penguins were more than 50 meters and two dives exceeded 240 meters. The at-sea metabolic rate, estimated from the turnover of tritiated water, was 2.8 times the standard metabolic rate and requires about 2.5 kilograms of squid per day. Ten percent or less of the dives may result in prey capture.