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A 4-base pair deletion in the mitochondrial cytochrome b gene associated with parkinsonism/MELAS overlap syndrome.

Five patients with diminished activity of complex III of the mitochondrial respiratory chain have been screened for mutations in the mitochondrial cytochrome b (cyt b) gene. In 1 patient, a young boy with an akinetic rigid syndrome and a mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), a novel 4-base pair deletion was identified. This mutation in this highly conserved gene is considered to be pathogenic since it is a heteroplasmic frame shift mutation predicted to lead to a truncated protein.

Adult↗

Saccharomyces cerevisiae YDR1, which encodes a member of the ATP-binding cassette (ABC) superfamily, is required for multidrug resistance.

A multidrug resistance gene, YDR1, of Saccharomyces cerevisiae, which encodes a 170-kDa protein of a member of the ABC superfamily, was identified. Disruption of YDR1 resulted in hypersensitivity to cycloheximide, cerulenin, compactin, staurosporine and fluphenazine, indicating that YDR1 is an important determinant of cross resistance to apparently-unrelated drugs. The Ydr1 protein bears the highest similarity to the S. cerevisiae Snq2 protein required for resistance to the mutagen 4-NQO. The drug-specificity analysis of YDR1 and SNQ2 by gene disruption, and its phenotypic suppression by the overexpressed genes, revealed overlapping, yet distinct, specificities. YDR1 was responsible for cycloheximide, cerulenin and compactin resistance, whereas, SNQ2 was responsible for 4-NQO resistance. The two genes had overlapping specificities toward staurosporine and fluphenazine. The transcription of YDR1 and SNQ2 was induced by various drugs, both relevant and irrelevant to the resistance caused by the gene, suggesting that drug specificity can be mainly attributed to the functional difference of the putative transporters. The transcription of these genes was also increased by heat shock. The yeast drug-resistance system provides a novel model for mammalian multidrug resistance.

ATP-Binding Cassette Transporters↗

Isolation and characterization of the mouse cardiac myosin heavy chain genes.

Two mouse genomic libraries were probed in order to isolate the murine cardiac myosin heavy chain (MHC) genes. Two overlapping cosmid clones that encode the cardiac genes were isolated. One of them encompasses the entire alpha-cardiac MHC gene, its 5'-flanking region and approximately 10 kilobase pairs (kb) of the 3'-end of the beta-cardiac MHC gene which we determined is located approximately 4 kb upstream of the alpha-cardiac MHC gene. Four clones isolated from a bacteriophage library were found to overlap with the 5'-region of the cosmid clones, and sequence analysis confirmed that the 5'-end of the beta-cardiac MHC gene was contained within one of the clones. Primer extension and polymerase chain reaction (PCR) analyses were used to define the transcriptional start site and the 5'-organization of the alpha-cardiac MHC gene. This region exhibits greater than 90% homology to the corresponding nucleotides of the rat alpha-cardiac MHC gene. To assess the importance of the intergenic region in directing expression of the alpha-cardiac MHC gene, a fragment containing the 3'-end of the beta-cardiac gene and the 5'-end of the alpha-cardiac gene was linked to a chloramphenicol acetyltransferase gene and used to generate transgenic mice. Analyses of the chloramphenicol acetyltransferase (CAT) activity in two lines indicate that the intergenic region is sufficient to properly direct expression in a tissue-specific manner.

Animals↗

Characterization of the Tupaia rhabdovirus genome reveals a long open reading frame overlapping with P and a novel gene encoding a small hydrophobic protein.

Rhabdoviruses are negative-stranded RNA viruses of the order Mononegavirales and have been isolated from vertebrates, insects, and plants. Members of the genus Lyssavirus cause the invariably fatal disease rabies, and a member of the genus Vesiculovirus, Chandipura virus, has recently been associated with acute encephalitis in children. We present here the complete genome sequence and transcription map of a rhabdovirus isolated from cultivated cells of hepatocellular carcinoma tissue from a moribund tree shrew. The negative-strand genome of tupaia rhabdovirus is composed of 11,440 nucleotides and encodes six genes that are separated by one or two intergenic nucleotides. In addition to the typical rhabdovirus genes in the order N-P-M-G-L, a gene encoding a small hydrophobic putative type I transmembrane protein of approximately 11 kDa was identified between the M and G genes, and the corresponding transcript was detected in infected cells. Similar to some Vesiculoviruses and many Paramyxovirinae, the P gene has a second overlapping reading frame that can be accessed by ribosomal choice and encodes a protein of 26 kDa, predicted to be the largest C protein of these virus families. Phylogenetic analyses of the tupaia rhabdovirus N and L genes show that the virus is distantly related to the Vesiculoviruses, Ephemeroviruses, and the recently characterized Flanders virus and Oita virus and further extends the sequence territory occupied by animal rhabdoviruses.

Amino Acid Sequence↗

Regulatory roles of conserved intergenic domains in vertebrate Dlx bigene clusters.

Dlx homeobox genes of vertebrates are generally arranged as three bigene clusters on distinct chromosomes. The Dlx1/Dlx2, Dlx5/Dlx6, and Dlx3/Dlx7 clusters likely originate from duplications of an ancestral Dlx gene pair. Overlaps in expression are often observed between genes from the different clusters. To determine if the overlaps are a result of the conservation of enhancer sequences between paralogous clusters, we compared the Dlx1/2 and the Dlx5/Dlx6 intergenic regions from human, mouse, zebrafish, and from two pufferfish, Spheroides nephelus and Takifugu rubripes. Conservation between all five vertebrates is limited to four sequences, two in Dlx1/Dlx2 and two in Dlx5/Dlx6. These noncoding sequences are >75% identical over a few hundred base pairs, even in distant vertebrates. However, when compared to each other, the four intergenic sequences show a much more limited similarity. Each intergenic sequence acts as an enhancer when tested in transgenic animals. Three of them are active in the forebrain with overlapping patterns despite their limited sequence similarity. The lack of sequence similarity between paralogous intergenic regions and the high degree of sequence conservation of orthologous enhancers suggest a rapid divergence of Dlx intergenic regions early in chordate/vertebrate evolution followed by fixation of cis-acting regulatory elements.

Animals↗

Molecular cloning, expression, and primary sequence of outer membrane protein P2 of Haemophilus influenzae type b.

The structural gene for the porin of Haemophilus influenzae type b, designated outer membrane protein P2, was cloned, and the DNA sequence was determined. An oligonucleotide probe generated by reverse translation of N-terminal amino acid sequence data from the purified protein was used to screen genomic DNA. The probe detected a single EcoRI fragment of approximately 1,700 base pairs which was cloned to lambda gt11 and then into M13 and partially sequenced. The derived amino acid sequence indicated that we had cloned the N-terminal portion of the P2 gene. An overlapping approximately 1,600-base-pair PvuII genomic fragment was cloned into M13, and the sequence of the remainder of the P2 gene was determined. The gene for P2 was then reconstructed under the control of the T7 promoter and expressed in Escherichia coli. The N-terminal sequence of the purified protein corresponds to residues 21 through 34 of the derived amino acid sequence. Thus, the protein is synthesized with a 20-amino-acid leader peptide. The Mr of the processed protein is 37,782, in good agreement with the estimate of 37,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Amino Acid Sequence↗

Characterization of the Pac25I restriction-modification genes isolated from the endogenous pRA2 plasmid of Pseudomonas alcaligenes NCIB 9867.

Genes for the class II Pseudomonas alcaligenes NCIB 9867 restriction-modification (R-M) system, Pac25I, have been cloned from its 33-kb endogenous plasmid, pRA2. The Pac25I endonuclease and methylase genes were found to be aligned in a head-to-tail orientation with the methylase gene preceding and overlapping the endonuclease gene by 1 bp. The deduced amino acid sequence of the Pac25I methylase revealed significant similarity with the XcyI, XmaI, Cfr9I, and SmaI methylases. High sequence similarity was displayed between the Pac25I endonuclease and the XcyI, XmaI, and Cfr9I endonucleases which cleave between the external cytosines of the recognition sequence (i.e., 5'-C CCGGG-3') and are thus perfect isoschizomers. However, no sequence similarity was detected between the Pac25I endonuclease and the SmaI endonuclease which cleaves between the internal CpG of the recognition sequence (i.e., 5'-CCCGGG-3'). Both the Pac25I methylase and endonuclease were expressed in Escherichia coli. An open reading frame encoding a protein which shows significant similarity to invertases and resolvases was located immediately upstream of the Pac25I R-M operon. In addition, a transposon designated Tn5563 was located 1531 bp downstream of the R-M genes. The location on a self-transmissible plasmid as well as the close association with genes involved in DNA mobility suggests horizontal transfer as a possible mode of distribution of this family of R-M genes in various bacteria.

Amino Acid Sequence↗

Nucleotide sequence and phylogenetic implication of the ATPase subunits beta and epsilon encoded in the chloroplast genome of the brown alga Dictyota dichotoma.

We have cloned and sequenced the genes atpB and atpE, coding for CF1 subunits beta and epsilon, respectively, of the chloroplast genome of the brown alga Dictyota dichotoma. Although the coding site of atpE cannot be demonstrated by heterologous Southern hybridizations, a 417 bp reading frame 3' to atpB was identified as the gene atpE by sequence similarities with atpE genes from other sources. A maximum sequence identity of 30% is found between the predicted amino acid sequence of the Dictyota subunit epsilon and the corresponding cyanobacterial subunits. Including conserved amino acid replacements, the Dictyota epsilon subunit exhibits about 70% sequence similarity with the cyanobacterial and land plant subunits. As in cyanobacteria, the atpE gene does not overlap the preceding gene atpB. The deduced amino acid sequence of atpB is 74-79% identical to the corresponding cyanobacterial and chloroplast subunits. Entirely conserved are regions referred to as the catalytic and/or regulatory sites of ATP formation, including interacting regions between subunits alpha and beta. A phylogram predicted from F1/CF1-beta subunits of eleven different organisms suggests a common evolutionary origin of plastids from chlorophytes and brown algae.

Amino Acid Sequence↗

Genomic organization and regulation of the vav proto-oncogene.

Vav and vav2 are members of the dbl family of guanine nucleotide exchange factors (GEF) for the rho/rac family of GTP binding proteins. Vav is expressed primarily in hematopoietic cells, while vav2 has a wider tissue distribution. The genomic structure of the human vav proto-oncogene was studied by identifying and sequencing all 27 exons of the gene from overlapping P1 and cosmid clones. The gene spans a 77-kb region on chromosome 19. In contrast, the coding region of vav2 is distributed over 30 exons spanning 227-kb. The overall organization of the exons which encode both proteins was found to be similar. In humans, alternative splicing of exons 6, 16 and 28 generated at least two distinct vav2 mRNA species. Several differences from the original vav cDNA sequence were noted. The most important difference was the identification of amino acid 718 as isoleucine, rather than threonine. This change warrants the reclassification of the vav SH2 domain as a type 3 SH2, instead of a type 2 SH2 as originally proposed by Songyang et al. (Mol. Cell. Biol. 14 (1994) 2777-2785). A series of vav promoter deletions were constructed using the enhanced green fluorescent protein (EGFP) as a reporter gene. A 23-bp segment that included a potential CBF/AML-1 binding site was found to be essential for EGFP expression in U937 cells. The same constructs were not active in HeLa cells, which do not express vav. A potential c-myb DNA binding site within the vav promoter was not required for EGFP expression.

3T3 Cells↗

An unusual mechanism of bacterial gene expression revealed for the RNase P protein of Thermus strains.

The RNase P protein gene (rnpA) completely overlaps the rpmH gene (encoding ribosomal protein L34) out of frame in the thermophilic bacterium Thermus thermophilus. This results in the synthesis of an extended RNase P protein (C5) of 163 aa and, by inference, of 240 aa in the related strain Thermus filiformis. Start codons of rnpA and rpmH, apparently governed by the same ribosome binding site, are separated by only 4 nt, which suggests a regulatory linkage between L34 and C5 translation and, accordingly, between ribosome and RNase P biosynthesis. Within the sequence encoding the N-terminal extensions and downstream of rpmH, several Thermus species exhibit in-frame deletionsinsertions, suggesting relaxed constraints for sequence conservation in this region. Roughly the N-terminal third of T. thermophilus C5 was further shown to be dispensable for RNase P function in vitro by using a precursor tRNA(Gly) substrate from the same organism. Taken together, these data reveal a mode of gene expression that is to our knowledge unprecedented in bacteria.

Amino Acid Sequence↗

Complete mitochondrial genome suggests diapsid affinities of turtles.

Despite more than a century of debate, the evolutionary position of turtles (Testudines) relative to other amniotes (reptiles, birds, and mammals) remains uncertain. One of the major impediments to resolving this important evolutionary problem is the highly distinctive and enigmatic morphology of turtles that led to their traditional placement apart from diapsid reptiles as sole descendants of presumably primitive anapsid reptiles. To address this question, the complete (16,787-bp) mitochondrial genome sequence of the African side-necked turtle (Pelomedusa subrufa) was determined. This molecule contains several unusual features: a (TA)n microsatellite in the control region, the absence of an origin of replication for the light strand in the WANCY region of five tRNA genes, an unusually long noncoding region separating the ND5 and ND6 genes, an overlap between ATPase 6 and COIII genes, and the existence of extra nucleotides in ND3 and ND4L putative ORFs. Phylogenetic analyses of the complete mitochondrial genome sequences supported the placement of turtles as the sister group of an alligator and chicken (Archosauria) clade. This result clearly rejects the Haematothermia hypothesis (a sister-group relationship between mammals and birds), as well as rejecting the placement of turtles as the most basal living amniotes. Moreover, evidence from both complete mitochondrial rRNA genes supports a sister-group relationship of turtles to Archosauria to the exclusion of Lepidosauria (tuatara, snakes, and lizards). These results challenge the classic view of turtles as the only survivors of primary anapsid reptiles and imply that turtles might have secondarily lost their skull fenestration.

Animals↗

Human CPR (cell cycle progression restoration) genes impart a Far- phenotype on yeast cells.

Regulated cell cycle progression depends on the proper integration of growth control pathways with the basic cell cycle machinery. While many of the central molecules such as cyclins, CDKs, and CKIs are known, and many of the kinases and phosphatases that modify the CDKs have been identified, little is known about the additional layers of regulation that impinge upon these molecules. To identify new regulators of cell proliferation, we have selected for human and yeast cDNAs that when overexpressed were capable of specifically overcoming G1 arrest signals from the cell cycle branch of the mating pheromone pathway, while still maintaining the integrity of the transcriptional induction branch. We have identified 13 human CPR (cell cycle progression restoration) genes and 11 yeast OPY (overproduction-induced pheromone-resistant yeast) genes that specifically block the G1 arrest by mating pheromone. The CPR genes represent a variety of biochemical functions including a new cyclin, a tumor suppressor binding protein, chaperones, transcription factors, translation factors, RNA-binding proteins, as well as novel proteins. Several CPR genes require individual CLNs to promote pheromone resistance and those that require CLN3 increase the basal levels of Cln3 protein. Moreover, several of the yeast OPY genes have overlapping functions with the human CPR genes, indicating a possible conservation of roles.

Amino Acid Sequence↗

Genie--gene finding in Drosophila melanogaster.

A hidden Markov model-based gene-finding system called Genie was applied to the genomic Adh region in Drosophila melanogaster as a part of the Genome Annotation Assessment Project (GASP). Predictions from three versions of the Genie gene-finding system were submitted, one based on statistical properties of coding genes, a second included EST alignment information, and a third that integrated protein sequence homology information. All three programs were trained on the provided Drosophila training data. In addition, promoter assignments from an integrated neural network were submitted. The gene assignments overlapped >90% of the 222 annotated genes and 26 possibly novel genes were predicted, of which some might be overpredictions. The system correctly identified the exon boundaries of 70% of the exons in cDNA-confirmed genes and 77% of the exons with the addition of EST sequence alignments. The best of the three Genie submissions predicted 19 of the annotated 43 gene structures entirely correct (44%). In the promoter category, only 30% of the transcription start sites could be detected, but by integrating this program as a sensor into Genie the false-positive rate could be dropped to 1/16,786 (0.006%). The results of the experiment on the long contiguous genomic sequence revealed some problems concerning gene assembly in Genie. The results were used to improve the system. We show that Genie is a robust hidden Markov model system that allows for a generalized integration of information from different sources such as signal sensors (splice sites, start codon, etc.), content sensors (exons, introns, intergenic) and alignments of mRNA, EST, and peptide sequences. The assessment showed that Genie could effectively be used for the annotation of complete genomes from higher organisms.

Animals↗

Repression of the LEAFY COTYLEDON 1/B3 regulatory network in plant embryo development by VP1/ABSCISIC ACID INSENSITIVE 3-LIKE B3 genes.

Plant embryo development is regulated by a network of transcription factors that include LEAFY COTYLEDON 1 (LEC1), LEC1-LIKE (L1L), and B3 domain factors, LEAFY COTYLEDON 2 (LEC2), FUSCA3 (FUS3), and ABSCISIC ACID INSENSITIVE 3 (ABI3) of Arabidopsis (Arabidopsis thaliana). Interactions of these genes result in temporal progression of overlapping B3 gene expression culminating in maturation and desiccation of the seed. Three VP1/ABI3-LIKE (VAL) genes encode B3 proteins that include plant homeodomain-like and CW domains associated with chromatin factors. Whereas val monogenic mutants have phenotypes similar to wild type, val1 val2 double-mutant seedlings form no leaves and develop embryo-like proliferations in root and apical meristem regions. In a val1 background, val2 and val3 condition a dominant variegated leaf phenotype revealing a VAL function in vegetative development. Reminiscent of the pickle (pkl) mutant, inhibition of gibberellin biosynthesis during germination induces embryonic phenotypes in val1 seedlings. Consistent with the embryonic seedling phenotype, LEC1, L1L, ABI3, and FUS3 are up-regulated in val1 val2 seedlings in association with a global shift in gene expression to a profile resembling late-torpedo-stage embryogenesis. Hence, VAL factors function as global repressors of the LEC1/B3 gene system. The consensus binding site of the ABI3/FUS3/LEC2 B3 DNA-binding domain (Sph/RY) is strongly enriched in the promoters and first introns of VAL-repressed genes, including the early acting LEC1 and L1L genes. We suggest that VAL targets Sph/RY-containing genes in the network for chromatin-mediated repression in conjunction with the PKL-related CHD3 chromatin-remodeling factors.

Arabidopsis↗

Nucleotide sequence of the simian virus 40 Hind-K restriction fragment.

The restriction fragment Hind-K represents 4.2% of the genome of Simian virus 40 (SV40) and is located near the middle of the late region. Its nucleotide sequence is reported here. It was mainly established by analysis of transcription products, synthesized by means of Escherichia coli RNA polymerase and nucleoside triphosphates, one of which was (alpha-32P)-labeled. Strand assignment was possible by hybridization of asymmetric, labeled transcripts of total SV40 DNA to filter-bound Hind-K fragment. Further information and unambiguous confirmation of the sequence was obtained by the use of direct DNA-sequencing methods. For this purpose the fragment was labeled at the 5' ends by means of polynucleotide kinase and [gamma-32P]ATP and redigested with a suitable restriction enzyme. The separated products were then either partially digested with snake venom diesterase for analysis by the 'wandering spot' method or partially degraded with the base-specific reagents dimethylsulphate or hydrazine for direct sequence analysis on gel. The Hind-K sequence is 219 base pairs long. The message strand is particularly rich in adenosine (39%) and purines. The nucleotide sequence cna unambiguously be translated into an amino acid sequence and the N-terminal codon of the viral protein VP1 gene could be identified. The amino-terminal part of VP1 is rich in proline and lysine. The nucleotide sequence of Hind-K codes also for the carboxyl-terminal part of the viral protein VP2 and VP3 genes, which partly overlap the VP1 gene.

Base Sequence↗

Genome-wide transcriptional profiling of the Escherichia coli responses to superoxide stress and sodium salicylate.

Escherichia coli responds to oxidative stress by activating sets of coregulated genes that help the cell to maintain homeostasis. Identified previously by genetic and biochemical approaches, the soxRS system mediates the induction of 18 of these redox-inducible genes (including the soxS gene itself). An overlapping set of genes is activated by an assortment of structurally unrelated molecules with antibiotic activities; many genes in this response are controlled by the marRAB system. The activation of either the soxRS or the marRAB system results in enhanced resistance to both superoxide-generating agents and multiple antibiotics. In order to probe the extent of these regulatory networks, we have measured whole-genome transcriptional profiles of the E. coli response to the superoxide-generating agent paraquat (PQ), an inducer of the soxRS system, and to the weak acid salt sodium salicylate (NaSal), an inducer of the marRA system. A total of 112 genes was modulated in response to PQ, while 134 genes were modulated in response to NaSal. We have also obtained transcriptional profiles of the SoxS and MarA regulons in the absence of global stress, in order to establish the regulatory hierarchies within the global responses. Several previously unrelated genes were shown to be under SoxS or MarA control. The genetic responses to both environmental insults revealed several common themes, including the activation of genes coding for functions that replenish reducing potential; regulate iron transport and storage; and participate in sugar and amino acid transport, detoxification, protein modification, osmotic protection, and peptidoglycan synthesis. A large number of PQ- and NaSal-responsive genes have no known function, suggesting that many adaptive metabolic changes that ensue after stress remain uncharacterized.

Bacterial Proteins↗

Comparative profiling of the sense and antisense transcriptome of maize lines.

BACKGROUND: There are thousands of maize lines with distinctive normal as well as mutant phenotypes. To determine the validity of comparisons among mutants in different lines, we first address the question of how similar the transcriptomes are in three standard lines at four developmental stages. RESULTS: Four tissues (leaves, 1 mm anthers, 1.5 mm anthers, pollen) from one hybrid and one inbred maize line were hybridized with the W23 inbred on Agilent oligonucleotide microarrays with 21,000 elements. Tissue-specific gene expression patterns were documented, with leaves having the most tissue-specific transcripts. Haploid pollen expresses about half as many genes as the other samples. High overlap of gene expression was found between leaves and anthers. Anther and pollen transcript expression showed high conservation among the three lines while leaves had more divergence. Antisense transcripts represented about 6 to 14 percent of total transcriptome by tissue type but were similar across lines. Gene Ontology (GO) annotations were assigned and tabulated. Enrichment in GO terms related to cell-cycle functions was found for the identified antisense transcripts. Microarray results were validated via quantitative real-time PCR and by hybridization to a second oligonucleotide microarray platform. CONCLUSION: Despite high polymorphisms and structural differences among maize inbred lines, the transcriptomes of the three lines displayed remarkable similarities, especially in both reproductive samples (anther and pollen). We also identified potential stage markers for maize anther development. A large number of antisense transcripts were detected and implicated in important biological functions given the enrichment of particular GO classes.

Expressed Sequence Tags↗

A DNA Motif Lexicon: cataloguing and annotating sequences.

The rapid proliferation of genomic DNA sequences has created a significant need for software that can both focus on relatively small areas (such as within genes or promoters) and provide wide-zoom views of patterns across entire genomes. We present our DNA Motif Lexicon that enables users to perform genome-wide searches for motifs of interest and create customizable results pages, where results differ in the degree and extent of annotation. Searching for a particular motif is akin to a word search in a natural language; our motif lexicon speaks to this new time when we will increasingly rely upon DNA dictionaries that offer rich types of annotation. Indeed, the concept of "lexomics", introduced in this paper may be appropriate to the types of meta-analyses appropriate to the deciphering of regulatory information. Currently supporting five genomes, our web-based lexicon allows users to look up motifs of interest and build user-defined result pages to include the following: (1) all base pair locations where a motif is found with links to further search the "neighborhoods" near each of these locations; whether each location of the motif is genic (within) a gene, intergenic, or a bridging sequence (overlapping a gene boundary) (2) NCBI hot-links to nearest upstream and downstream genes for each location (3) statistical information about the query (4) whether the motif is a certain type of repeat (5) links for the reverse, complement and reverse-complement of the motif of interest and (6) hot-links to PubMed abstracts which mention the motif of interest. A software framework facilitates the continual development of new annotation modules. The tool is located at: http://genomics.wheatoncollege.edu/cgi-bin/lexicon.exe.

Animals↗