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The genomics of yeast responses to environmental stress and starvation.

Unicellular organisms such as yeast have evolved to survive constant fluctuations in their external surroundings by rapidly adapting their internal systems to meet the challenges of each new environment. One aspect of this cellular adaptation is the reorganization of genomic expression to the program required for growth in each environment. The reprogramming of genomic expression can be unveiled using DNA microarrays, which measure the relative transcript abundance of essentially every gene in an organism's genome. Characterizing environmentally triggered gene expression changes provides insights into when, where, and how each gene is expressed and offers a glimpse at the physiological response of the cells to changes in their surroundings. This review will focus on the genomic expression responses of the budding yeast Saccharomyces cerevisiae to diverse environmental changes, highlighting some of the themes that have emerged from the collection of published yeast genomic expression studies. The results of these studies present insights as to how yeast cells sense and respond to each new environment, and suggest mechanisms that this organism uses to survive stressful environmental changes.

Culture Media↗

Structure and expression analysis of early auxin-responsive Aux/IAA gene family in rice (Oryza sativa).

Auxin exerts pleiotropic effects on plant growth and development by regulating the expression of early auxin-responsive genes of auxin/indoleacetic acid (Aux/IAA), small auxin-up RNA, and GH3 classes. These genes have been studied extensively in dicots like soybean and Arabidopsis. We had earlier characterized a cDNA of the first monocot member of Aux/IAA family from rice. The achievement of the large scale rice genome sequencing combined with the availability of full-length cDNA sequences from Knowledge-based Oryza Molecular Biological Encyclopedia provided us the opportunity to draw up the first comprehensive list of Aux/IAA genes in a monocot. By screening the available databases, we have identified 31 Aux/IAA genes having high sequence identity within the conserved domains I, II, III, and IV. The genomic organization as well as chromosomal location of all the Oryza sativa indoleacetic acid (OsIAA) genes is reported. The rice Aux/IAA proteins can be classified in two groups (A and B) on the basis of their phylogenetic relationship with Arabidopsis Aux/IAA proteins. An evolutionary pattern of the rice Aux/IAA genes has been discussed by analyzing their structure (exon/intron organization) and duplications. Interestingly, the duplication of rice Aux/IAA genes was found to be associated with chromosomal block duplication events in rice. The in-silico analysis has been complemented with real-time polymerase chain reaction analysis to quantify transcript levels of all Aux/IAA family members. OsIAA genes showed differential and overlapping organ-specific expression patterns in light- and dark-grown seedlings/plants. Although auxin enhanced the transcript abundance of most of the OsIAA genes, the effect was more pronounced on OsIAA9, 14, 19, 20, 24, and 31. These results provide a foundation for future studies on elucidating the precise role of rice Aux/IAA genes in early steps of auxin signal transduction.

Amino Acid Sequence↗

The auxin-responsive GH3 gene family in rice (Oryza sativa).

Auxin regulates plant growth and development by altering the expression of diverse genes. Among these, the genes of Aux/IAA, SAUR, and GH3 classes have been extensively studied in dicots, but little information is available on monocots. We have identified 12 members of GH3 gene family in rice using sequences of full-length cDNA clones available from KOME and analysis of the whole genome sequence of rice. The genomic organization as well as chromosomal location of all the OsGH3 genes is reported. The rice GH3 proteins can be classified in two groups (groups I and II) on the basis of their phylogenetic relationship with Arabidopsis GH3 proteins. Based upon the sequences available in the database, not a single group III GH3 protein could be identified in rice. An extensive survey of EST sequences of other monocots led to the conclusion that although GH3 gene family is highly conserved in both dicots and monocots but the group III is conspicuous by its absence in monocots. The in silico analysis has been complemented with experimental data to quantify transcript levels of all GH3 gene family members. Using real-time polymerase chain reaction, the organ-specific expression of individual OsGH3 genes in light- and dark-grown seedlings/plants has been examined. The transcript abundance of nearly all OsGH3 genes is enhanced on auxin treatment, with the effect more pronounced on OsGH3-1, -2, and -4. The functional validation of these genes in transgenics or analysis of gene-specific insertional mutants will help in elucidating their precise role in auxin signal transduction.

Amino Acid Sequence↗

Gene discovery in the auditory system: characterization of additional cochlear-expressed sequences.

To identify genes involved in hearing, 8494 expressed sequence tags (ESTs) were generated from a human fetal cochlear cDNA library in two distinct sequencing projects. Analysis of the first set of 4304 ESTs revealed clones representing 517 known human genes, 41 mammalian genes not previously detected in human tissues, 487 ESTs from other human tissues, and 541 cochlear-specific ESTs (http://hearing.bwh.harvard.edu). We now report results of a DNA sequence similarity (BLAST) analysis of an additional 4190 cochlear ESTs and a comparison to the first set. Among the 4190 new cochlear ESTs, 959 known human genes were identified; 594 were found only among the new ESTs and 365 were found among ESTs from both sequencing projects. COL1A2 was the most abundant transcript among both sets of ESTs, followed in order by COL3A1, SPARC, EEFY1A1, and TPTI. An additional 22 human homologs of known nonhuman mammalian genes and 1595 clusters of ESTs, of which 333 are cochlear-specific, were identified among the new cochlear ESTs. Map positions were determined for 373 of the new cochlear ESTs and revealed 318 additional loci. Forty-nine of the mapped ESTs are located within the genetic interval of 23 deafness loci. Reanalysis of unassigned ESTs from the prior study revealed 338 additional known human genes. The total number of known human genes identified from 8494 cochlear ESTs is 1449 and is represented by 4040 ESTs. Among the known human genes are 14 deafness-associated genes, including GJB2 (connexin 26) and KVLQT1. The total number of nonhuman mammalian genes identified is 43 and is represented by 58 ESTs. The total number of ESTs without sequence similarity to known genes is 4055. Of these, 778 also do not have sequence similarity to any other ESTs, are categorized into 700 clusters, and may represent genes uniquely or preferentially expressed in the cochlea. Identification of additional known genes, ESTs, and cochlear-specific ESTs provides new candidate genes for both syndromic and nonsyndromic deafness disorders.

Auditory Pathways↗

Microarray analyses of the metabolic responses of Saccharomyces cerevisiae to organic solvent dimethyl sulfoxide.

The toxic effects that organic solvents have on whole cells are important drawbacks in the application of these solvents in the production of fine chemicals by whole-cell stereoselective biotransformations. Although early studies found that organic solvents mainly destroyed the integrity of cell membranes by accumulating in the lipid bilayer of plasma membranes, the cellular metabolic responses to the presence of an organic solvent remain unclear. With the rapid development of genomics, it is possible to study cellular metabolism under perturbed conditions at the genome level. In this paper, the global gene expression profiles of Saccharomyces cerevisiae BY4743 grown in media with a high concentration of the organic solvent dimethyl sulfoxide (DMSO) were determined by microarray analysis of ~6,200 yeast open reading frames (ORFs). From cells grown in SD minimal medium containing 1.0% (v/v) DMSO, changes in transcript abundance greater than or equal to 2.5-fold were classified. Genomic analyses showed that 1,338 genes were significantly regulated by the presence of DMSO in yeast. Among them, only 400 genes were previously found to be responsive to general environmental stresses, such as temperature shock, amino acid starvation, nitrogen source depletion, and progression into stationary phase. The DMSO-responsive genes were involved in a variety of cellular functions, including carbohydrate, amino acid and lipid metabolism, cellular stress responses, and energy metabolism. Most of the genes in the lipid biosynthetic pathways were down-regulated by DMSO treatment, whereas genes involved in amino acid biosynthesis were mostly up-regulated. The results demonstrate that the application of microarray technology allows better interpretation of metabolic responses, and the information obtained will be useful for the construction of engineered yeast strains with better tolerance of organic solvents.

Dimethyl Sulfoxide↗

Temporal evolution of the Arabidopsis oxidative stress response.

We have carried out a detailed analysis of the changes in gene expression levels in Arabidopsis thaliana ecotype Columbia (Col-0) plants during and for 6 h after exposure to ozone (O3) at 350 parts per billion (ppb) for 6 h. This O3 exposure is sufficient to induce a marked transcriptional response and an oxidative burst, but not to cause substantial tissue damage in Col-0 wild-type plants and is within the range encountered in some major metropolitan areas. We have developed analytical and visualization tools to automate the identification of expression profile groups with common gene ontology (GO) annotations based on the sub-cellular localization and function of the proteins encoded by the genes, as well as to automate promoter analysis for such gene groups. We describe application of these methods to identify stress-induced genes whose transcript abundance is likely to be controlled by common regulatory mechanisms and summarized our findings in a temporal model of the stress response.

Arabidopsis↗

Proliferation-associated changes in in vitro mRNA translation in the HL60 cell line.

In order to characterize patterns of gene expression during the proliferation cycle of HL60 cells, we have analysed changes in the population of mRNA available for translation in vitro. HL60 cells were separated into cell cycle phases by centrifugal elutriation, monitoring the separation with flow cytometry. RNA was extracted from cell fractions highly enriched in G1, S or G2+M phases and translated in vitro. Translation products were analysed by two-dimensional electrophoresis followed by autoradiography. Autoradiograms were analysed by a computer-assisted method utilizing a drum-scanning microdensitometer. Spots were identified by their relative positions on the films and their relative intensity was estimated. Of the 159 peptides studied for cell cycle-associated changes in synthesis, nine showed phase-associated changes. The most significant changes were the accumulation of four peptides that showed maximal synthesis only in G2+M phases. An additional four peptides were synthesized maximally in both S and G2+M phases. One peptide showed maximal synthesis in S phase. These changes in gene expression suggest that these relatively abundant transcripts are regulated primarily at a quantitative level during proliferation and may be related to the doubling of structural proteins prior to mitosis.

Cell Line↗

Xenopus laevis oocyte G alpha subunits mRNAs. Detection and quantitation during oogenesis and early embryogenesis by competitive reverse PCR.

The expression of mRNAs coding for different Xenopus laevis oocyte G alpha subunits was analyzed by the PCR technique. Using the nucleotide sequences of five previously cloned cDNAs for oocyte G alpha subunits [FEBS Lett. 244, 188-192, 1989; FEBS Lett. 268, 27-31, 1990] and the highly sensitive reverse PCR reaction we found that G alpha o, G alpha i-1, G alpha i-3 and G alpha s species are present in oocyte stage VI, G alpha o mRNA being the most abundant transcript. G alpha o mRNA was further quantitated through oogenesis, unfertilized eggs and early embryogenesis stages by a competitive PCR reaction using an 'in vitro' deleted G alpha o mRNA as the internal standard. Using this approach we found that Xenopus G alpha o mRNA levels were constant during oogenesis and unfertilized eggs at a concentration of 3.5 pg of mRNA/stage (5 x 10(5) molecules) and diminish gradually during early embryogenesis, reaching a level of 0.3 pg in the gastrula stage. These findings show that oocyte G alpha o, and perhaps the rest of the alpha subunits, are expressed as maternal mRNAs and could play an important role in signal transduction at the beginning of oocyte cell differentiation.

Animals↗

Coexpression of the human papillomavirus type 16 E4 and L1 open reading frames in early cervical neoplasia.

Although the E4 open reading frame (ORF) of human papillomaviruses (HPV) encodes an abundant protein in cutaneous warts, the location and extent of HPV E4 expression in genital precancers, specifically those associated with HPV-16, has not been described. Expression plasmids (pATH) containing segments of the HPV-16 E4 (3401-3620) and L1 (6151-6792) open reading frames (ORFs) were induced and expressed in bacteria and the resulting fusion proteins were used to elicit antisera in rabbits. Antisera reacting to the E4 and L1 components of the fusion proteins were used to screen biopsies from 150 cervical precancers (cervical intraepithelial neoplasia) and condylomata. Six biopsies exhibiting specific immunostaining with the anti-E4 sera. Staining was cytoplasmic, and occurred virtually always in foci containing immunostaining for L1 proteins. Moreover, analysis of these 6 cases and 22 others for HPV-16 RNA by RNA-RNA in situ hybridization demonstrated a similar correlation between E4 immunostaining and the presence of abundant transcripts specific to HPV-16. These data are consistent with the hypothesis that expression of the HPV-16 E4 ORF is dependent upon viral replication and epithelial differentiation, similar to L1 expression, and that the E4 epitopes identified by the rabbit antisera may be unique to HPV-16 relative to other common cervical papillomaviruses.

Condylomata Acuminata↗

Human papillomavirus type 16 and 18 gene expression in cervical neoplasias.

Human papillomavirus (HPV) types 16 and 18 are strongly implicated in the generation of progressive cervical neoplasms. The viruses produce complex families of overlapping messenger RNAs that are linked to differentiation, making it necessary to analyze gene expression in the context of morphology. We have developed HPV type 16 and type 18 subgenomic clones from which 3H-labeled riboprobes specific to individual mRNA families can be generated in vitro. Using these probes for in situ hybridization, we examined serial sections of archival biopsy specimens of the spectrum of genital lesions. In low-grade squamous lesions, all viral open reading frames were expressed, and the most abundant transcription spanned the E4 and E5 open reading frames at the 3' end of the E region. L region transcription coding for the capsid proteins was restricted to terminally differentiated keratinocytes. As the grade of neoplasia increased, cellular differentiation and overall viral transcription decreased and, with few exceptions, the L2 and L1 transcripts ceased to exist. The E6-E7 transforming region was invariably derepressed. Interestingly, the patterns of HPV-16 gene expression suggested the coexistence of episomal and integrated viral DNAs. In contrast, in HPV-18 lesions, all the viral template DNA appeared to have integrated. Integration was deduced to have occurred near the boundary of the E1 and E2 open reading frames. Viral transcription patterns were similar in carcinomas in situ and in invasive carcinomas, regardless of the histologic cell types or the associated virus types, consistent with the notion that additional host gene alterations were necessary for progression. On the basis of viral gene expression in vivo and the E6 promoter regulation previously characterized in vitro, we discuss a molecular mechanism for HPV-associated carcinogenesis.

Adenocarcinoma↗

The human papillomavirus type 16 E7 gene encodes transactivation and transformation functions similar to those of adenovirus E1A.

Clinical and epidemiological data have implicated the human papillomaviruses (HPVs) as having an etiologic role in some anogenital malignancies, with HPV-16 being most frequently (greater than 60%) detected in cervical carcinoma. HPV-16 is actively transcribed in the cancers; the most abundant transcripts map to the E6 and E7 early open reading frames. Evidence is presented that the HPV-16 E7 open reading frame encodes transcriptional transactivation and cellular transformation functions analogous to those of adenovirus E1A proteins. Specifically, the HPV-16 E7 gene product could transactivate the adenovirus E2 promoter and cooperate with an activated ras oncogene to transform primary baby rat kidney cells. The E7 transforming function differed somewhat from that of adenovirus E1A in that E7 was also able to transform established mouse cells. Examination of the amino acid sequence of HPV-16 E7 revealed striking similarities with conserved domains 1 and 2 of adenovirus E1A proteins.

Adenoviruses, Human↗

Temporal and spatial pattern of cellular myc oncogene expression during human placental development.

The human placental trophoblast component is of embryonic origin and is developmentally regulated; the tissue is highly proliferative and often described as pseudomalignant. Because cellular oncogenes have been implicated in normal cellular proliferation and differentiation processes, we have studied c-myc oncogene expression in relation to the progression of human placental development. The c-myc transcript shows a 20- to 30-fold variation over the course of placental development, with a peak at four to five weeks after conception. A clear decline in placental c-myc transcription is seen before the end of the first trimester of pregnancy. In situ hybridization to [125I]-labelled myc probes demonstrates an unequal distribution of myc transcripts in placenta, with particularly high expression in the cytotrophoblastic shell of the early placenta. The localization of myc transcripts to cytotrophoblast and the temporal pattern of myc expression support a strong correlation between myc transcript abundance and cytotrophoblastic proliferation. These findings are discussed in the light of a possible role for the c-myc gene in proliferation of normal cells.

Female↗

Simultaneous use of digoxigenin- and radiolabeled oligodeoxyribonucleotide probes for hybridization histochemistry.

Oligodeoxyribonucleotide probes have proven very useful for studying gene expression in tissue sections, especially from the central nervous system. We report here a procedure for simultaneously identifying two different mRNA species in the same sections by hybridizing one with a digoxigenin-labeled probe, the other with a radiolabeled probe. The former probe is located by alkaline phosphatase-conjugated antibodies against digoxigenin, the latter by conventional nuclear emulsion autoradiography. Although the digoxigenin detection scheme currently lacks the sensitivity of autoradiography, it offers greater rapidity, resolution, and ease of detection of abundant transcripts. Furthermore, changes in gene expression within a subset of characterized cells is now feasible.

Animals↗

Regulated splicing of the amyloid precursor protein gene during postnatal development of the rat basal forebrain.

The expression of the amyloid precursor protein (APP) gene has been examined in the basal forebrain of rats from birth to adulthood. Levels of total APP mRNA are highest at birth and at postnatal day 15 (P15). The most abundant transcript in rat brain is APP-695, whose expression has previously been found to be largely restricted to the central nervous system. Comparison of the developmental profiles of APP-695 mRNA with that of Kunitz-protease inhibitor (KPI)-containing APP mRNA shows that the greatest difference in expression occurs at P15, when APP-695 message levels are over 6-fold higher than KPI-containing APP mRNA (APP-751, APP-770). This is the largest difference in the APP-695/KPI-APP ratio observed during postnatal development and coincides with the period of maximal neurotrophic responsiveness in the basal forebrain. These results suggest that the APP gene is alternatively spliced during postnatal development and that regulated expression of APP-695 may be influenced by neurotrophic factors in vivo.

Amyloid beta-Protein Precursor↗

Characterization of a Giardia lamblia variant-specific surface protein (VSP) gene from isolate GS/M and estimation of the VSP gene repertoire size.

Giardia lamblia undergoes surface antigenic variation. The variant-specific surface proteins (VSPs) of isolate WB are cysteine-rich, can vary dramatically in size, contain Cys-X-X-Cys motifs, and are differentially expressed. GS/M(H7) is a Giardia clone from a different isolate which expresses a VSP epitope not found in WB. The VSP gene encoding this epitope was selected by differential hybridization using radiolabeled cDNA from H7 and variant sibling trophozoite lines from the same isolate that express other VSPs. The VSPH7 gene probe detects an 1800 nucleotide transcript abundant in H7 but undetectable in variant siblings. Primer extension directly from RNA was used to complete the gene sequence which predicted a protein with a molecular weight of 56,832. The protein showed many of the characteristics of 2 previously sequenced WB VSPs including many Cys-X-X-Cys tetrapeptides and a conserved carboxy-terminal region. Genomic Southern analysis indicated the presence of 2 distinct VSPH7 genes in H7. An oligonucleotide from the conserved region was used in combination with one specific for the VSPH7 gene to estimate the VSP repertoire size at between 133 and 151. VSPs, even from isolates expressing unique epitopes, constitute a family of related proteins.

Amino Acid Sequence↗

Nucleotide sequence of the most abundantly transcribed early gene of human cytomegalovirus strain AD169.

Cytoplasmic poly(A+)RNA was isolated from human embryo fibroblast cells during the early phase of an infection with human cytomegalovirus strain AD169. These preparations contained a single abundant transcript of 2.7 kb which was derived from each of the repeat sequences flanking the long unique region of the virus genome. The gene was unspliced and poly(A+)RNA derived from it continued to accumulate in the cytoplasm of cells during the later stages of infection. This gene and the surrounding region was sequenced. A potential open reading frame of 170 amino acids was identified close to the 5'-terminus of the gene; the 2.7 kb early transcript therefore contains a long untranslated 3'-sequence. The promoter sequences of the 2.7 kb early gene show homology with corresponding regions of the HSV-1 gD and the human hsp 70 genes.

Amino Acid Sequence↗

Expression of interferon-gamma from hybrid yeast GPD promoters containing upstream regulatory sequences from the GAL1-GAL10 intergenic region.

The expression of human immune interferon (IFN-gamma) is toxic to yeast, resulting in low plasmid stability and copy number. The Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene (GPD) promoter [Bitter and Egan, Gene 32 (1984) 263-274] has been modified by introduction of upstream regulatory sequences from the yeast GAL1-GAL10 intergenic region [UASG; Guarente et al., Proc. Natl. Acad. Sci. USA 79 (1982) 7410-7414] and utilized to express IFN-gamma. In contrast to the native GPD promoter, the GPD(G) hybrid promoters are regulated by the carbon source. With glucose as the carbon source, a level of expression is observed which is much lower than that obtained with the native GPD promoter. Expression of the hybrid promoters is induced approx. 150- to 200-fold in shaker flask cultures by growth in galactose and similar levels of expression are observed after growth in lactate plus galactose. However, full galactose induction is not observed in the presence of glucose.? Utilization of these regulated promoters has allowed maintenance of plasmids at high copy number with glucose as the carbon source and, after induction with galactose, production of IFN-gamma mRNA at levels more than ten times higher than the native yeast PGK gene transcript. In contrast, the native GPD promoter directs comparable levels of expression when grown in either glucose or galactose resulting in low plasmid copy number and a correspondingly lower IFN-gamma transcript abundance. It is demonstrated that nucleotide sequences more than 240 bp upstream from the TATA box are required for optimal activity of the native GPD promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning, Molecular↗

Isolation of Trichoderma reesei genes highly expressed on glucose-containing media: characterization of the tef1 gene encoding translation elongation factor 1 alpha.

Genes that are highly expressed on glucose-containing media were isolated from the filamentous fungus, Trichoderma reesei. A cDNA bank was prepared from glucose-grown fungus, the bank was screened with the same cDNA as a probe, and clones giving the strongest signal were isolated. This resulted in the isolation of previously uncharacterized genes. Five of the genes, representing the most abundant transcripts, corresponded to 1-3% of the total mRNA population and were clearly more highly expressed than the phosphoglycerate kinase-encoding gene (pgk1) of T. reesei. Based on sequence homology, one of the genes was identified as tef1, encoding translation elongation factor 1 alpha (TEF). The T. reesei TEF is most related to the Mucor racemosus TEF3, showing an overall amino acid similarity of 85%. Interestingly, an exon of only 2 bp seems to be present in T. reesei tef1, comprising the first 2 bp of the Gly15 codon.

Amino Acid Sequence↗