Sequence of a cytochrome c gene from Kluyveromyces lactis and its upstream region

The complete sequence of a cytochrome c gene from Kluyveromyces lactis including its upstream region is reported. Sequence of the translated open reading frame is discussed in terms of cytochrome c structural requirements. Putative regulatory signals in the upstream region are described and compared with reported sequences which modulate the expression of respiratory-related yeast genes.     

Transcriptional control of AAC3 gene encoding mitochondrial ADP/ATP translocator in Saccharomyces cerevisiae by oxygen, heme and ROX1 factor

The AAC3 gene of Saccharomyces cerevisiae encodes a mitochondrial ADP/ATP translocator which is subject to oxygen repression. Evidence is presented here, that the repression of AAC3 expression is dependent upon heme and the ROX1 factor. The promoter region of the AAC3 gene was isolated, sequenced, and deletion analysis was performed using lacZ as a reporter gene to determine the cis-acting regions responsible for the regulation of AAC3 expression. The results of the deletion analysis show that the negative control of the AAC3 gene by oxygen and ROX1 factor is mediated by an upstream repression sequence consisting of a T-rich segment adjacent to the consensus elements that are present in the 5' flanking regions of several other yeast genes. An additional upstream repressor site was located within the AAC3 promoter which, however, is not related either to oxygen or to ROX1 factor. The data presented here delineate the main cellular factors and DNA sequences involved in the regulatory mechanism by which an essential function for anaerobic cells growth, ADP/ATP translocation, is ensured. In addition, they show that the AAC3 gene belongs to the family of yeast genes including TIF51B, COX5b, HEM13 and CYC7 that are negatively regulated by oxygen and heme.     

Towards Arabidopsis genome analysis: monitoring expression profiles of 1400 genes using cDNA microarrays

cDNA microarrays containing 1443 Arabidopsis thaliana genes were analyzed for expression profiles in major organs of Arabidopsis plants. Novel expression profiles were identified for many coding sequences with putative gene identifications. Expression patterns of novel sequences provided clues to their possible functions. The results demonstrate how microarrays containing a large number of Arabidopsis genes can provide a powerful tool for plant gene discovery, functional analysis and elucidation of genetic regulatory networks.     

Phosphorylation of serum response factor by casein kinase II: evidence against a role in growth factor regulation of fos expression

With the sequencing of the first complete eukaryotic chromosome, III of yeast (YCIII) of length 315 kb, several types of questions concerning chromosomal organization and the heterogeneity of eukaryotic DNA sequences can be approached. We have undertaken extensive analysis of YCIII with the goals of: (1) discerning patterns and anomalies in the occurrences of short oligonucleotides; (2) characterizing the nature and locations of significant direct and inverted repeats; (3) delimiting regions unusually rich in particular base types (e.g., G+C, purines); and (4) analyzing the distributions of markers of interest, e.g., delta (delta) elements, ARS (autonomous replicating sequences), special oligonucleotides, close repeats and close dyad pairings, and gene sequences. YCIII reveals several distinctive sequence features, including: (i) a relative abundance of significant local and global repeats highlighting five genes containing substantial close or tandem DNA repeats; (ii) an anomalous distribution of delta elements involving two clusters and a long gap; (iii) a significantly even distribution of ARS; (iv) a relative increase in the frequency of T runs and AT iterations downstream of genes and A runs upstream of genes; and (v) two regions of complex repetitive sequences and anomalous DNA composition, 29000-31000 and 291000-295000, the latter centered at the HMRa locus. Interpretations of these findings for chromosomal organization and implications for regulation of gene expression are discussed.     

Map positions of 47 Arabidopsis sequences with sequence similarity to disease resistance genes

Map positions have been determined for 42 non-redundant Arabidopsis expressed sequence tags (ESTs) showing similarity to disease resistance genes (R-ESTs), and for three Pto-like sequences that were amplified with degenerate primers. Employing a PCR-based strategy, yeast artificial chromosome (YAC) clones containing the EST sequences were identified. Since many YACs have been mapped, the locations of the R-ESTs could be inferred from the map positions of the YACs. R-EST clones that exhibited ambiguous map positions were mapped as either cleavable amplifiable polymorphic sequence (CAPS) or restriction fragment length polymorphism (RFLP) markers using F8 (Ler x Col-0) recombinant inbred (RI) lines. In all cases but two, the R-ESTs and Pto-like sequences mapped to single, unique locations. One R-EST and one Pto-like sequence each mapped to two locations. Thus, a total of 47 loci were identified in this study. Several R-ESTs occur in clusters suggesting that they may have arisen via gene duplication events. Interestingly, several R-ESTs map to regions containing genetically defined disease resistance genes. Thus, this collection of mapped R-ESTs may expedite the isolation of disease resistance genes. As the cDNA sequencing projects have identified an estimated 63% of Arabidopsis genes, a very large number of R-ESTs (approximately 95), and by inference disease resistance genes of the leucine-rich repeat-class probably occur in the Arabidopsis genome.     

Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications

A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes.     

Analysis of EST-driven gene annotation in human genomic sequence

We have performed a systematic analysis of gene identification in genomic sequence by similarity search against expressed sequence tags (ESTs) to assess the suitability of this method for automated annotation of the human genome. A BLAST-based strategy was constructed to examine the potential of this approach, and was applied to test sets containing all human genomic sequences longer than 5 kb in public databases, plus 300 kb of exhaustively characterized benchmark sequence. At high stringency, 70%-90% of all annotated genes are detected by near-identity to EST sequence; >95% of ESTs aligning with well-annotated sequences overlap a gene. These ESTs provide immediate access to the corresponding cDNA clones for follow-up laboratory verification and subsequent biologic analysis. At lower stringency, up to 97% of annotated genes were identified by similarity to ESTs. The apparent false-positive rate rose to 55% of ESTs among all sequences and 20% among benchmark sequences at the lowest stringency, indicating that many genes in public database entries are unannotated. Approximately half of the alignments span multiple exons, and thus aid in the construction of gene predictions and elucidation of alternative splicing. In addition, ESTs from multiple cDNA libraries frequently cluster over genes, providing a starting point for crude expression profiles. Clone IDs may be used to form EST pairs, and particularly to extend models by associating alignments of lower stringency with high-quality alignments. These results demonstrate that EST similarity search is a practical general-purpose annotation technique that complements pattern recognition methods as a tool for gene characterization.     

Genomic disorders: structural features of the genome can lead to DNA rearrangements and human disease traits

Molecular medicine began with Pauling's seminal work, which recognized sickle-cell anemia as a molecular disease, and with Ingram's demonstration of a specific chemical difference between the hemoglobins of normal and sickled human red blood cells. During the four decades that followed, investigations have focused on the gene--how mutations specifically alter DNA and how these changes affect the structure and expression of encoded proteins. Recently, however, the advances of the human genome project and the completion of total genome sequences for yeast and many bacterial species, have enabled investigators to view genetic information in the context of the entire genome. As a result, we recognize that the mechanisms for some genetic diseases are best understood at a genomic level. The evolution of the mammalian genome has resulted in the duplication of genes, gene segments and repeat gene clusters. This genome architecture provides substrates for homologous recombination between nonsyntenic regions of chromosomes. Such events can result in DNA rearrangements that cause disease.     

A carbon source-responsive promoter element necessary for activation of the isocitrate lyase gene ICL1 is common to genes of the gluconeogenic pathway in the yeast Saccharomyces cerevisiae

The expression of yeast genes encoding gluconeogenic enzymes depends strictly on the carbon source available in the growth medium. We have characterized the control region of the isocitrate lyase gene ICL1, which is derepressed more than 200-fold after transfer of cells from fermentative to nonfermentative growth conditions. Deletion analysis of the ICL1 promoter led to the identification of an upstream activating sequence element, UASICL1 (5' CATTCATCCG 3'), necessary and sufficient for conferring carbon source-dependent regulation on a heterologous reporter gene. Similar sequence motifs were also found in the upstream regions of coregulated genes involved in gluconeogenesis. This carbon source-responsive element (CSRE) interacts with a protein factor, designated Ang1 (activator of nonfermentative growth), detectable only in extracts derived from derepressed cells. Gene activation mediated by the CSRE requires the positively acting derepression genes CAT1 (= SNF1 and CCR1) and CAT3 (= SNF4). In the respective mutants, Ang1-CSRE interaction was no longer observed under repressing or derepressing conditions. Since binding of Ang1 factor to the CSRE could be competed for by an upstream sequence derived from the fructose-1,6-bisphosphatase gene FBP1, we propose that the CSRE functions as a UAS element common to genes of the gluconeogenic pathway.     

Complete sequence of a hair-like intermediate filament type II keratin gene

The Intermediate Filament (IF) superfamily comprises several multigene families, of which the two keratin families are the largest. The keratin IF genes are expressed in epithelial tissues in differentiation-specific patterns and recently we reported the sequence and expression of a hair IF type II keratin gene (KRT2.9). Two related genes were present in the cosmid containing KRT2.9 and we have now sequenced one of them and found that it encodes a hair-like IF type II protein (KRT2.13). However, KRT2.13 is not expressed in the hair follicle. Interestingly there is significant sequence homology between introns 1, 5 and 6 of KRT2.13 and KRT2.9 to suggest gene conversion of these regions or possibly conservation of functional sequences.     

Genotoxicity and oxidative stress induced by pesticide exposure in bovine lymphocyte cultures in vitro

Pectin demethylesterification appears to be catalysed by a number of pectin methylesterase (PME) isoenzymes in higher plant species. In order to better define the biological role of these isoenzymes in plant cell growth and differentiation, we undertook molecular studies on the PME-encoding genes in Arabidopsis thaliana. In this paper, we report the characterization of AtPME3, a new PME-related gene of 4kb in length that we have mapped on Chromosome III. AtPME3 encodes a putative mature PME-related isoenzyme of 34kDa with a basic isoelectric point. Since the extent of the gene family encoding PME in higher plant species is still unknown, we resorted to the use of degenerate primers designed from several well-known consensus regions to identify new PME-related genes in the genome of Arabidopsis. Our results, in combination with several known expressed sequences tags (ESTs), indicate that the Arabidopsis genome contains at least 12 PME-related genes. Consequently, a method of systematic gene expression analysis has been applied in order to discern the expression pattern of these 12 genes throughout the plant at the floral stage. Whereas most of these genes appeared to be more or less ubiquitously expressed throughout the plant, several genes are distinguishable by their strikingly specific expression in certain organs. The present data bring a new insight into the role of specific PME-related genes in flower and root development.