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Results for “Genomic characterization”

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PubMed1992

The P-glycoprotein gene family of Caenorhabditis elegans. Cloning and characterization of genomic and complementary DNA sequences.

P-glycoproteins, encoded by families of evolutionarily conserved genes, can confer a multidrug-resistant phenotype to mammalian tumor cells. To obtain more information on their functions in normal cells we have cloned genomic and complementary DNA sequences of four P-glycoprotein gene homologs of the genetically well-characterized nematode Caenorhabditis elegans, termed pgp-1, pgp-2, pgp-3 and pgp-4, respectively. The genes were physically mapped on chromosome IV (pgp-1), I (pgp-2) and X (pgp-3 and pgp-4). Phenotypic mutants corresponding to these loci have not yet been described. Two of the genes, pgp-1 and pgp-3, were analyzed in detail. They are predicted to encode ATP-binding membrane-spanning proteins of 1321 and 1254 amino acid residues, respectively, with the characteristic features shared by most P-glycoproteins described thus far. Intra-species divergence of P-glycoprotein genes is more pronounced in C. elegans than in mammals. Only 40% of the amino acids of pgp-1 and pgp-3 are identical, in contrast to 77% identity between human MDR1 and MDR3. pgp-1 consists of 14 exons, pgp-3 of 13. The two genes share only one intron position, whereas they share four (pgp-1) and five (pgp-3) intron positions with mammalian P-glycoprotein genes. pgp-1, pgp-2, and pgp-3 are transcribed into low abundance mRNAs in wild-type nematodes. pgp-1 and pgp-3 mRNAs have the trans-spliced leader SL1 at their 5' ends. Arsenite, emetine and actinomycin D drugs did not increase the steady state levels of pgp mRNA, unlike in some mammalian cell types. Heat shock disturbed trans as well as cis-splicing of pgp-1 and led to the accumulation of partially processed pgp-1 RNA. Thus, in C. elegans these genes are not induced in the context of a general stress response, as has been proposed for mammalian P-glycoprotein genes in certain tissues.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

Microviridae, a family divided: isolation, characterization, and genome sequence of phiMH2K, a bacteriophage of the obligate intracellular parasitic bacterium Bdellovibrio bacteriovorus.

A novel single-stranded DNA phage, phiMH2K, of Bdellovibrio bacteriovorus was isolated, characterized, and sequenced. This phage is a member of the Microviridae, a family typified by bacteriophage phiX174. Although B. bacteriovorus and Escherichia coli are both classified as proteobacteria, phiMH2K is only distantly related to phiX174. Instead, phiMH2K exhibits an extremely close relationship to the Microviridae of Chlamydia in both genome organization and encoded proteins. Unlike the double-stranded DNA bacteriophages, for which a wide spectrum of diversity has been observed, the single-stranded icosahedral bacteriophages appear to fall into two distinct subfamilies. These observations suggest that the mechanisms driving single-stranded DNA bacteriophage evolution are inherently different from those driving the evolution of the double-stranded bacteriophages.

Amino Acid Sequence↗

PubMed2000

Characterization and genomic localization of the mouse Extl2 gene.

Human EXTL2 is an alpha1,4-N-acetylhexosaminyltransferase involved in the biosynthesis of heparin/heparan sulfate. We have cloned and characterized the mouse homolog of this gene. Mouse Extl2 encodes a 330 amino acid protein that is 87% identical to its human counterpart. Expression analysis showed that Extl2 is ubiquitously expressed in adult mouse tissues and that the Extl2 transcript is already present in early stages of embryonic development. Determination of the genomic structure revealed that the Extl2 gene spans five exons within a 10-kb region and that the genomic organization between mouse and man is well preserved, with conservation of the number and position of all five exons. By radiation hybrid analysis, Extl2 was mapped to mouse chromosome 3, in a region homologous to the human EXTL2 region on chromosome 1.

Amino Acid Sequence↗

PubMed1994

A genetic selection method for the transfer of HSV-1 glycoprotein B mutations from plasmid to the viral genome: preliminary characterization of transdominance and entry kinetics of mutant viruses.

A genetic selection procedure has been devised to transfer mutations from a plasmid to the viral genome. Reagents were constructed so that the recombination events that occur during cotransfection rescue a mutation in a recipient viral genome and, simultaneously, transfer a mutation in an adjacent (target) gene to the viral genome. In the example presented here the two adjacent genes are essential for viral replication, so that a transformed cell line that expresses both genes and another cell line that expresses only the target gene are required. Ideally the recipient viral genome should be deleted for the entire target gene, and the deletion should extend a short distance into the adjacent gene. In the present study UL27 (glycoprotein gB) of herpes simplex virus type 1 (KOS) is the target gene for mutation transfer and the upstream gene UL28, which specifies the ICP18.5 polypeptide, is the marker-rescue gene. A recipient virus that was deleted for DNA sequences encoding the C-terminal 74 residues of ICP18.5 and the N-terminal 711 residues of gB was constructed. All of the gB mutant plasmids used overlap the ICP18.5 deletion, and therefore, recombination events that rescue the ICP18.5 deletion must transfer a gB mutation present in codons 1-711 of the rescuing plasmid. Recombinant viruses that contain the gB mutation, unlike the parental gB-/18.5- virus, will grow on cells that express only gB (D6). This procedure has been used to transfer insertion, deletion, and chain termination mutations into the gB gene of the KOS genome. Southern blot analysis confirmed the transfer of the mutations to the viral genome. Analysis of radioactively labeled, infected cell lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), before and after immune precipitation with gB-specific antibodies, confirmed the presence of the mutant polypeptides in the lysates. The incorporation of the mutant polypeptides into the envelopes of purified released viruses was measured. The yield of plaque forming units per cell was determined from single step growth curves after infection of permissive D6 cells. Virus yield, a measure of transdominance, varied for the different mutants. The maximum reduction in virus yield observed was sixfold relative to the wild-type virus. Transdominance was a result of both the lack of glycoprotein processing and the presence of gB-oligomerization sites. A reduced yield was also observed for a previously isolated gB-null mutant virus, K082. The amount of gB produced in D6 cells, infected with K082, was less than that produced in KOS infected cells, which may account for the reduced yield.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Line↗

PubMed2001

Euteleost fish genomes are characterized by expansion of gene families.

The presence of additional hox clusters in the zebrafish has led to the hypothesis that there was a whole genome duplication at the origin of modern fish. To investigate the generality of this assumption, we analyzed all available actinopterygian fish gene families, and sequenced nuclear receptors from diverse teleost fish. The origin and timing of duplications was systematically determined by phylogenetic analysis. More genes are indeed found in zebrafish than in mouse. This abundance is shared by all major groups of euteleost fish, but not by eels. Phylogenetic analysis shows that it may result from frequent independent duplications, rather than from an ancestral genome duplication. We predict two zebrafish paralogs for most mouse or human genes, thus expressing a note of caution in functional comparison of fish and mammalian genomes. Redundancy appears to be the rule in fish developmental genetics. Finally, our results imply that the outcome of genome projects cannot be extrapolated easily between fish species.

Animals↗

PubMed2000

Genome-wide characterization of the Zap1p zinc-responsive regulon in yeast.

The Zap1p transcription factor senses cellular zinc status and increases expression of its target genes in response to zinc deficiency. Previously known Zap1p-regulated genes encode the Zrt1p, Zrt2p, and Zrt3p zinc transporter genes and Zap1p itself. To allow the characterization of additional genes in yeast important for zinc homeostasis, a systematic study of gene expression on the genome-wide scale was used to identify other Zap1p target genes. Using a combination of DNA microarrays and a computer-assisted analysis of shared motifs in the promoters of similarly regulated genes, we identified 46 genes that are potentially regulated by Zap1p. Zap1p-regulated expression of seven of these newly identified target genes was confirmed independently by using lacZ reporter fusions, suggesting that many of the remaining candidate genes are also Zap1p targets. Our studies demonstrate the efficacy of this combined approach to define the regulon of a specific eukaryotic transcription factor.

Gene Expression Profiling↗

PubMed1988

Characterization of genomic clones coding for the C alpha and C beta subunits of mouse cAMP-dependent protein kinase.

Mouse genes for two isozymes of the catalytic subunit of cAMP-dependent protein kinase (C alpha, C beta) were isolated and characterized. The C alpha gene is divided into 10 exons contained on three overlapping clones. Genomic clones containing the 5' end of the C beta gene were also isolated, and sequence analysis demonstrated that the positions of the three introns examined are identical in the C alpha and C beta genes. S1 mapping and primer extension experiments indicate that transcription initiated from multiple sites in both genes and that neither contain recognizable TATA or CAAT homologies. The 5' flanking regions of both genes are remarkably high in GC content (80% for C alpha and 72% for C beta) and are similar to the "HTF" (HpaII tiny fragment fraction) island sequences which have recently been found to flank several other mammalian transcriptional units.

Amino Acid Sequence↗

PubMed2003

Genome-wide characterization of the lignification toolbox in Arabidopsis.

Lignin, one of the most abundant terrestrial biopolymers, is indispensable for plant structure and defense. With the availability of the full genome sequence, large collections of insertion mutants, and functional genomics tools, Arabidopsis constitutes an excellent model system to profoundly unravel the monolignol biosynthetic pathway. In a genome-wide bioinformatics survey of the Arabidopsis genome, 34 candidate genes were annotated that encode genes homologous to the 10 presently known enzymes of the monolignol biosynthesis pathway, nine of which have not been described before. By combining evolutionary analysis of these 10 gene families with in silico promoter analysis and expression data (from a reverse transcription-polymerase chain reaction analysis on an extensive tissue panel, mining of expressed sequence tags from publicly available resources, and assembling expression data from literature), 12 genes could be pinpointed as the most likely candidates for a role in vascular lignification. Furthermore, a possible novel link was detected between the presence of the AC regulatory promoter element and the biosynthesis of G lignin during vascular development. Together, these data describe the full complement of monolignol biosynthesis genes in Arabidopsis, provide a unified nomenclature, and serve as a basis for further functional studies.

Alcohol Oxidoreductases↗

PubMed1991

Characterization and genomic organization of a highly expressed late nodulin gene subfamily in soybeans.

A soybean nodulin cDNA clone (E41) hybrid-selected mRNA for three in vitro translation products with apparent molecular weights of 26 kDa, 25 kDa and 24 kDa. Based on Southern analysis of soybean genomic DNA, combined with mapping and sequencing of genomic clones, we identified four genes that are related to E41, one of which was identified to be the previously characterized N-20 gene. Our data indicate the linkage of three of the genes, of which one is a truncated version and suggest that they originated by gene duplication combined with deletion and conversion. The genes are highly expressed and we postulate that the sequence conservation in the 5' and 3' flanking regions of all four genes, has a functional role in their expression. Hybrid-selected translation products of E41 are not immunoprecipitable with antibody to the soluble fraction of nodules suggesting that they are membrane associated. The N-20 gene, which is a member of this gene subfamily, showed sequence similarity to four previously characterized nodulin genes and a phylogenetic tree is proposed based on the extent of sequence similarity.

Base Sequence↗

PubMed2006

Genome-wide characterization of fission yeast DNA replication origins.

Eukaryotic DNA replication is initiated from multiple origins of replication, but little is known about the global regulation of origins throughout the genome or in different types of cell cycles. Here, we identify 401 strong origins and 503 putative weaker origins spaced in total every 14 kb throughout the genome of the fission yeast Schizosaccharomyces pombe. The same origins are used during premeiotic and mitotic S-phases. We found that few origins fire late in mitotic S-phase and that activating the Rad3 dependent S-phase checkpoint by inhibiting DNA replication had little effect on which origins were fired. A genome-wide analysis of eukaryotic origin efficiencies showed that efficiency was variable, with large chromosomal domains enriched for efficient or inefficient origins. Average efficiency is twice as high during mitosis compared with meiosis, which can account for their different S-phase lengths. We conclude that there is a continuum of origin efficiency and that there is differential origin activity in the mitotic and meiotic cell cycles.

Chromosomes, Fungal↗

PubMed2000

Characterization and genomic sequence of the murine 60 kD Ro gene.

Autoantibodies binding 60 kD Ro (or SS-A) are commonly found in patients with systemic lupus erythematosus and Sjögren's syndrome. While many studies have examined the autoimmune response directed against this RNA-protein, its function is still uncertain. As part of a broad effort to better understand animal models of anti-Ro autoimmunity we have characterized the murine 60 kD Ro gene. Southern blot analysis of mouse genomic DNA suggests that the 60 kD Ro gene is a single copy gene. The complete sequence of the gene was determined from three overlapping genomic lambda phage clones (GenBank accession number AF065398). The murine 60 kD Ro gene spans approximately 23 kb and consists of 8 or 9 exons. DNA sequence analysis revealed the presence of multiple B1 repetitive units. It maps in synteny with the human 60 kD Ro gene. Therefore, the isolation and characterization of the 60 kD Ro gene will be instrumental for future studies on protein function and the role this protein plays in the development of autoimmune responses.

Animals↗

PubMedDate not supplied

Molecular characterization of genome of a novel human T-cell leukaemia virus.

A novel human retrovirus (HTLV-II) was previously found associated with a T-cell variant of hairy-cell leukaemia. Molecular cloning demonstrates that the complete provirus genome is 8.8 kilobase pairs in size and is transmissible to uninfected cells. Two types of infectious deleted provirus were also characterized. The sequences of HTLV-II are distinct from those of HTLV-I.

Base Sequence↗

PubMed2005

Genome-wide characterization of gene expression variations and DNA copy number changes in prostate cancer cell lines.

BACKGROUND: The aim of this study was to characterize gene expression and DNA copy number profiles in androgen sensitive (AS) and androgen insensitive (AI) prostate cancer cell lines on a genome-wide scale. METHODS: Gene expression profiles and DNA copy number changes were examined using DNA microarrays in eight commonly used prostate cancer cell lines. Chromosomal regions with DNA copy number changes were identified using cluster along chromosome (CLAC). RESULTS: There were discrete differences in gene expression patterns between AS and AI cells that were not limited to androgen-responsive genes. AI cells displayed more DNA copy number changes, especially amplifications, than AS cells. The gene expression profiles of cell lines showed limited similarities to prostate tumors harvested at surgery. CONCLUSIONS: AS and AI cell lines are different in their transcriptional programs and degree of DNA copy number alterations. This dataset provides a context for the use of prostate cancer cell lines as models for clinical cancers.

Cell Line, Tumor↗

PubMed2026

Rice LSD1-like Genes: Genome-Wide Characterization and Evidence Linking OsLSD3 to Plant Height.

LSD1-like zinc-finger proteins participate in programmed cell death, redox homeostasis, and stress responses in plants, but their functional diversification and contributions to agronomic variation in rice remain poorly defined. This study aimed to characterize the rice LSD1-like gene family and evaluate the potential agronomic roles of selected members, with particular emphasis on OsLSD3. Genome-wide analyses were integrated with OsLSD3 natural variation and haplotype analyses in 4666 rice accessions, CRISPR/Cas9 mutant phenotyping in the ZH11 background, and subcellular localization assays. Seven LSD1-like genes were identified and showed substantial divergence in protein architecture, gene organization, promoter cis-element profiles, and tissue- and stress-responsive expression. OsLSD3 formed six population-structured haplotypes, and two common Japonica haplotypes differed significantly in plant height. Consistently, two independent oslsd3 mutant lines were taller than the wild type, whereas additional changes in grain-related traits were line-specific. OsLSD2 and OsLSD3 localized mainly to the nucleus, while OsLSD4 was predominantly nuclear with weak cytoplasmic localization. These results identify OsLSD3 as the strongest candidate among the examined members for further investigation of plant height- and grain-related traits, while OsLSD2 and OsLSD4 represent additional candidates for grain-trait regulation. Further validation using additional alleles and environments is required.

LSD1-like↗

PubMed2005

Gene identification signature (GIS) analysis for transcriptome characterization and genome annotation.

We have developed a DNA tag sequencing and mapping strategy called gene identification signature (GIS) analysis, in which 5' and 3' signatures of full-length cDNAs are accurately extracted into paired-end ditags (PETs) that are concatenated for efficient sequencing and mapped to genome sequences to demarcate the transcription boundaries of every gene. GIS analysis is potentially 30-fold more efficient than standard cDNA sequencing approaches for transcriptome characterization. We demonstrated this approach with 116,252 PET sequences derived from mouse embryonic stem cells. Initial analysis of this dataset identified hundreds of previously uncharacterized transcripts, including alternative transcripts of known genes. We also uncovered several intergenically spliced and unusual fusion transcripts, one of which was confirmed as a trans-splicing event and was differentially expressed. The concept of paired-end ditagging described here for transcriptome analysis can also be applied to whole-genome analysis of cis-regulatory and other DNA elements and represents an important technological advance for genome annotation.

5' Flanking Region↗

PubMed2026

Mannheimia haemolytica strain-level diversity in cattle populations.

High-resolution genomic characterization is essential for understanding diversity, pathogenicity, and transmission dynamics of bacterial pathogens. Mannheimia haemolytica (Mh) is the most consequential bacterial agent associated with bovine respiratory disease (BRD) in cattle, as a leading cause of morbidity, mortality, and antimicrobial use. Historically, BRD pathogens, including Mh, have been studied using culture or PCR approaches that provided limited ability to characterize fine-scale genomic variation across communities. Here, we evaluated target-enriched (TE) shotgun sequencing, a culture-independent method capable of strain-level resolution within metagenomic data, for detecting and characterizing Mh in comparison with qPCR and 16S rRNA gene sequencing. Nasal swabs (10 individual and 2 composited DNA samples per pen) and environmental samples (three ropes hung on pen rails and three water bowl swabs per pen) were collected from four pens in each of five distinct cattle populations. DNA was extracted for TE sequencing to identify Mh at both species and genomic sequence variant (GSV) levels, and to characterize antimicrobial resistance genes across the bacterial communities. qPCR was performed to quantify Mh genome copies, and 16S rRNA gene sequencing was used to assess the broader respiratory microbiome. TE sequencing identified Mh in 100% of TE-tested samples and classified multiple GSVs in all but 3 of 121 samples. GSV profiles clustered within housing groups and varied across cattle populations, indicating structured strain-level diversity. In contrast, Mannheimia spp. were detected in only 47.7% of samples by 16S rRNA sequencing. These findings demonstrate that TE sequencing enables sensitive, strain-level characterization of Mh in cattle and environmental samples and reveals substantial within-population genomic diversity not captured by conventional approaches.IMPORTANCETarget-enriched shotgun sequencing enabled sensitive, strain-level detection of Mannheimia haemolytica (Mh), revealing multiple co-circulating genomic sequence variants (GSVs) within and among cattle groups. This demonstrates greater genetic variability of Mh populations in beef cattle than has been previously recognized. The clustering of GSVs within housing groups, together with the overlap between respiratory and environmental samples, is consistent with the hypothesis that contagious transmission contributes to Mh ecology. These results highlight the potential utility of composite nasal swab and environmental samples for future studies evaluating relationships between Mh genomic variation and disease risk.

Animals↗

PubMed1999

Molecular characterization and genome organization of 7SL RNA genes from hop (Humulus lupulus L.).

A wide spectrum of hop 7SL RNA-encoding sequences was detected by temperature gradient-gel electrophoresis. Four hop 7SL RNA genes were cloned and characterized. A new subvariant of the upstream sequence element (USE) 5'TCCCACATGG 3' and two distinct variants of TATA signal were found at positions characteristic for RNA polymerase III-driven transcription in plants. In addition, a more distant conserved sequence element 5' CATGTATAAACTTTCTGC 3' was present in all cloned genes, about 160 bp upstream of the 7SL RNA coding sequence. Consensus secondary structures calculated for hop 7SL RNAs revealed characteristic features, although some structure differences from formerly published models were predicted. Specific in-vitro transcription of plant 7SL RNA genes was observed in a heterologous system (HeLa extract). This in-vitro transcription assay showed significant differences among individual clones in transcription rates, suggesting the requirement of complexity of 7SL RNA sequence for its efficient transcription in HeLa extract. Southern blot analysis of hop DNA revealed 12 7SL-specific signals corresponding to HindIII fragments ranging from 0.45 to 7.8 kb. Several 7SL RNA-encoding sequences and various intergenic spacers were amplified from the individual HindIII fragments of about 1.3 and 2.8 kb. These facts suggest that at least some of the hop 7SL RNA genes are organized in genomic clusters.

Base Sequence↗

PubMed1996

Characterization and genomic mapping of genes and pseudogenes of a new human protein tyrosine phosphatase.

Previously described protein tyrosine phosphatases (PTPs) are classified into three types according to their sequence homology and structural features. Here we describe the characterization of genes and pseudogenes of a member of a fourth type of PTP, designated protein tyrosine phosphatase 4A (PTP4A). The 167-amino-acid human PTP4A bears the signature active site of all PTPs, but does not show any other sequence homology to any of the previously described PTPs. Two cDNAs encoding PTP4A that differed in their noncoding regions were isolated. Another cDNA that has a high level of sequence identity with these two cDNAs and a deletion in the coding region was also isolated. Northern analysis using a probe from a common 3'-untranslated region of the cDNAs recognized mRNAs of about 2 and 4 kb. Both species of mRNA were seen in all human adult and fetal tissues tested. Fluorescence in situ hybridization mapping of the corresponding yeast artificial chromosome clones and sequence-tagged site analysis suggested that one of the PTP4A coding genes is located at 1p35 and the other is on chromosome 11. A processed pseudogene for PTP4A was found in the BRCA1 region of 17q21 and shares 96% sequence identity to one of the PTP4A coding cDNAs. Our studies also suggest the existence of another processed pseudogene on chromosome 11.

Adult↗