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At least 199 records · Page 11Linked to original sources

PubMed2001

Molecular characterization of a highly divergent HIV type 1 isolate obtained early in the AIDS epidemic from the Democratic Republic of Congo.

Numerous complete human immunodeficiency virus type 1 (HIV-1) genomes have been characterized for contemporary viruses, but few isolates obtained early in the HIV-1 epidemic have been studied. In this article, we describe the molecular characterization of an HIV-1 isolate (83CD003) that was obtained from an AIDS patient in Kinshasa, Democratic Republic of Congo (DRC) in 1983. The complete 83CD003 genome was sequenced in its entirety and found to encode uninterrupted open reading frames for all viral genes. Phylogenetic analysis revealed 83CD003 was a member of the major (M) group of HIV -1, but did not group with any of the known subtypes. Rather, it formed an independent lineage in all regions of its genome that was roughly equidistant from representatives of all other subtypes. Similarly, 83CD003 also did not cluster with any of several unclassified group M sequences that have been reported more recently to circulate in the DRC, suggesting that it may represent an early group M lineage thai is either rare or has gone extinct. The molecular clone of 83CD003 yielded an infectious virus after transfection into mammalian cells and its biological properties can be further studied.

Cell Line↗

PubMed1998

Human gap junction protein connexin31: molecular cloning and expression analysis.

We have isolated and characterized a human genomic clone containing the complete coding region of connexin31 (Cx31). Similar to rodent Cx31, the coding region of human Cx31 is completely contained within the second exon and consists of 810 nucleotides. The deduced human Cx31 polypeptide consists of 270 amino acids with a predicted molecular mass of 30.818 kDa. Its sequence is most similar to mouse Cx31 (82.6% identical amino acids) and rat (83.0% identical amino acids), but shows considerably fewer potential sites of phosphorylation. After Northern blot hybridization, two Cx31 transcripts of 2.2 and 1.8 kb were detected in total RNA of the human keratinocyte cell line HaCaT and two transcripts of 2.2 and 1.9 kb in total RNA of E6/E7 transfected human keratinocytes (HEK cells). Using affinity-purified rabbit antibodies to mouse Cx31, immunofluorescence analysis demonstrated relatively weak expression of human Cx31 in HaCaT and HEK cells. The Cx31 gene exists as a single copy gene in the human genome and was mapped to the chromosomal region 1p34-p36 by analyzing human-mouse somatic cell hybrids.

Amino Acid Sequence↗

PubMed2000

Isolation and characterization of a class I SLG gene from Chinese cabbage (Brassica campestris).

In Brassica species, self-incompatibility in the recognition reaction between self and non-self pollens is determined by two genes, SLG and SRK, at the S locus. We have cloned and characterized a genomic DNA fragment containing a complete open reading frame of the SLG gene from Chinese cabbage. The genomic clone, named BcSLG2, was found to possess the region that shares a homology of 77% in amino acid identity with the SLG46 gene of Brassica campestris. Northern blot analysis revealed that the BcSLG2 gene expression is restricted to the pistil of Chinese cabbage flower. In situ hybridization showed that in the pistil, the gene is expressed predominantly in the stigmatic tissue. Much lower expression in the tapetum was also detectable at an immature stage of the flower development. Southern blot hybridization with the BcSLG2 DNA probe showed polymorphism in the SLG gene organization of the Chinese cabbage plants. These results will provide valuable information in understanding the S gene complex of the Chinese cabbage plants.

Amino Acid Sequence↗

PubMed2000

Molecular characterization of methylmalonate semialdehyde dehydrogenase deficiency.

Three patients have been reported with (putative) methylmalonic semialdehyde dehydrogenase (MMSDH) deficiency. The urine metabolic pattern was strikingly different in all, including beta-alanine, 3-hydroxypropionic acid, both isomers of 3-amino- and 3-hydroxyisobutyric acids in one and 3-hydroxyisobutyric and lactic acids in a second, and mild methylmalonic aciduria in a third patient. In an effort to clarify these disparate metabolite patterns, we completed the cDNA structure, and characterized the genomic structure of human MMSDH gene in order to undertake molecular analysis. Only the first patient had alterations in the MMSDH coding region, revealing homozygosity for a 1336G > A transversion, which leads to substitution of arginine for highly conserved glycine at amino acid 446. No abnormalities of the MMSDH cDNA were detected in the other patients. These data provide the first molecular characterization of an inborn error of metabolism specific to the L-valine catabolic pathway.

Aldehyde Oxidoreductases↗

PubMed1998

Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence.

Countless millions of people have died from tuberculosis, a chronic infectious disease caused by the tubercle bacillus. The complete genome sequence of the best-characterized strain of Mycobacterium tuberculosis, H37Rv, has been determined and analysed in order to improve our understanding of the biology of this slow-growing pathogen and to help the conception of new prophylactic and therapeutic interventions. The genome comprises 4,411,529 base pairs, contains around 4,000 genes, and has a very high guanine + cytosine content that is reflected in the biased amino-acid content of the proteins. M. tuberculosis differs radically from other bacteria in that a very large portion of its coding capacity is devoted to the production of enzymes involved in lipogenesis and lipolysis, and to two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation.

Chromosome Mapping↗

PubMed2001

Molecular characterization of Bombyx mori cytoplasmic polyhedrosis virus genome segment 4.

The complete nucleotide sequence of the genome segment 4 (S4) of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) was determined. The 3,259-nucleotide sequence contains a single long open reading frame which spans nucleotides 14 to 3187 and which is predicted to encode a protein with a molecular mass of about 130 kDa. Western blot analysis showed that S4 encodes BmCPV protein VP3, which is one of the outer components of the BmCPV virion. Sequence analysis of the deduced amino acid sequence of BmCPV VP3 revealed possible sequence homology with proteins from rice ragged stunt virus (RRSV) S2, Nilaparvata lugens reovirus S4, and Fiji disease fijivirus S4. This may suggest that plant reoviruses originated from insect viruses and that RRSV emerged more recently than other plant reoviruses. A chimeric protein consisting of BmCPV VP3 and green fluorescent protein (GFP) was constructed and expressed with BmCPV polyhedrin using a baculovirus expression vector. The VP3-GFP chimera was incorporated into BmCPV polyhedra and released under alkaline conditions. The results indicate that specific interactions occur between BmCPV polyhedrin and VP3 which might facilitate BmCPV virion occlusion into the polyhedra.

Amino Acid Sequence↗

PubMed1995

The human lens intrinsic membrane protein MP70 (Cx50) gene: clonal analysis and chromosome mapping.

We have isolated and characterized a human genomic clone containing the complete coding region of lens intrinsic membrane protein MP70 (Cx50). The coding region of this DNA is completely contained within one exon, as is common of all connexins investigated to date. The size of the Cx50 coding region, from the initiating ATG to the terminating TGA is 1,299 nucleotides, coding for a polypeptide of 432 amino acids and having a translated molecular weight of 48,171 daltons. This Cx50 coding region DNA was used as a probe to analyze a panel of Southern blots of human-Chinese hamster somatic cell hybrid DNAs to assign the gene coding for Cx50 to its human chromosome. Control human and Chinese hamster DNAs displayed a distinct Eco R1 restriction fragment pattern when hybridized with the human Cx50 DNA probe. When somatic cell hybrid DNAs were restricted with Eco R1 and Southern blots hybridized with the human Cx50 DNA probe, the characteristic human restriction pattern was observed only when human chromosome 1 was present in the hybrid panel. Of the other six connexin genes which have previously been assigned to a human chromosome, two of these, Cx37 and Cx40, are also found on chromosome 1.

Amino Acid Sequence↗

PubMed1993

DNA sequence, structure and gene expression of mycobacteriophage L5: a phage system for mycobacterial genetics.

Genetic studies of Mycobacterium tuberculosis and other mycobacterial pathogens have suffered from the lack of a sophisticated genetic system. To address this issue we have developed a viral system through a detailed characterization of mycobacteriophage L5, a temperate phage that infects both fast- and slow-growing mycobacteria. We describe here the complete DNA sequence of the L5 genome and initial characterization of L5 virion structure and gene expression. In addition to providing a genetic 'tool-box' for the mycobacteria we find that L5 offers a new paradigm for dsDNA phages, being phenotypically temperate but employing genetic strategies for phage growth usually associated with lytic bacteriophages.

Amino Acid Sequence↗

PubMed2004

Transcriptomic and proteomic characterization of the Fur modulon in the metal-reducing bacterium Shewanella oneidensis.

The availability of the complete genome sequence for Shewanella oneidensis MR-1 has permitted a comprehensive characterization of the ferric uptake regulator (Fur) modulon in this dissimilatory metal-reducing bacterium. We have employed targeted gene mutagenesis, DNA microarrays, proteomic analysis using liquid chromatography-mass spectrometry, and computational motif discovery tools to define the S. oneidensis Fur regulon. Using this integrated approach, we identified nine probable operons (containing 24 genes) and 15 individual open reading frames (ORFs), either with unknown functions or encoding products annotated as transport or binding proteins, that are predicted to be direct targets of Fur-mediated repression. This study suggested, for the first time, possible roles for four operons and eight ORFs with unknown functions in iron metabolism or iron transport-related functions. Proteomic analysis clearly identified a number of transporters, binding proteins, and receptors related to iron uptake that were up-regulated in response to a fur deletion and verified the expression of nine genes originally annotated as pseudogenes. Comparison of the transcriptome and proteome data revealed strong correlation for genes shown to be undergoing large changes at the transcript level. A number of genes encoding components of the electron transport system were also differentially expressed in a fur deletion mutant. The gene omcA (SO1779), which encodes a decaheme cytochrome c, exhibited significant decreases in both mRNA and protein abundance in the fur mutant and possessed a strong candidate Fur-binding site in its upstream region, thus suggesting that omcA may be a direct target of Fur activation.

Bacterial Proteins↗

PubMed1998

Determination of the genomic structure of the COL4A4 gene and of novel mutations causing autosomal recessive Alport syndrome.

Autosomal recessive Alport syndrome is a progressive hematuric glomerulonephritis characterized by glomerular basement membrane abnormalities and associated with mutations in either the COL4A3 or the COL4A4 gene, which encode the alpha3 and alpha4 type IV collagen chains, respectively. To date, mutation screening in the two genes has been hampered by the lack of genomic structure information. We report here the complete characterization of the 48 exons of the COL4A4 gene, a comprehensive gene screen, and the subsequent detection of 10 novel mutations in eight patients diagnosed with autosomal recessive Alport syndrome. Furthermore, we identified a glycine to alanine substitution in the collagenous domain that is apparently silent in the heterozygous carriers, in 11.5% of all control individuals, and in one control individual homozygous for this glycine substitution. There has been no previous finding of a glycine substitution that is not associated with any obvious phenotype in homozygous individuals.

Alanine↗

PubMed2003

Calcium/calmodulin-mediated signal network in plants.

Various extracellular stimuli elicit specific calcium signatures that can be recognized by different calcium sensors. Calmodulin, the predominant calcium receptor, is one of the best-characterized calcium sensors in eukaryotes. In recent years, completion of the Arabidopsis genome project and advances in functional genomics have helped to identify and characterize numerous calmodulin-binding proteins in plants. There are some similarities in Ca(2+)/calmodulin-mediated signaling in plants and animals. However, plants possess multiple calmodulin genes and many calmodulin target proteins, including unique protein kinases and transcription factors. Some of these proteins are likely to act as "hubs" during calcium signal transduction. Hence, a better understanding of the function of these calmodulin target proteins should help in deciphering the Ca(2+)/calmodulin-mediated signal network and its role in plant growth, development and response to environmental stimuli.

Arabidopsis↗

PubMed1996

High-resolution FISH of the entire integrated Epstein-Barr virus genome on extended human DNA.

Here we report a high-resolution fluorescence in situ hybridization (FISH) analysis of the integrated Epstein-Barr virus (EBV) genome in chromosomes, decondensed interphase nuclear chromatin, and linearly extended chromatin fibers. We analyzed the EBV DNA integrated into the human genome in the well-characterized Burkitt's lymphoma cell line Namalwa, which contains two complete EBV genomes. The integration occurs via the terminal repeats of the virus and was always detectable at chromosome band 1p35. Using the biotinylated BamHIW fragment of the viral DNA, we observed distinct pairs of signals or small nuclear RNA "tracks" within interphase nuclei. FISH to stretched DNA fibers has a higher resolving power and; therefore, enables analysis of the structural organization of DNA. Application of this methodology to linearly extended chromatin of Namalwa cells using different EBV fragments allowed us to visualize the ordered arrangement of the integrated virus. Based on the predicted span of 0.34 nm per base pair for relaxed DNA, length measurements of 30 images showed a good correlation between the mean physical length of hybridized EBV DNA of 52.8 microns (158 kb) without the terminal repeats, and the EBV genomic length of 172 kb, including the terminal repeats. This DNA mapping procedure represents a useful tool for studying the structural organization of integrated viral genomes, and its application will have implications for the understanding of integration processes.

Burkitt Lymphoma↗

PubMed2006

Molecular characterization of the equine collagen, type IX, alpha 2 (COL9A2) gene on horse chromosome 2p16-->p15.

The mammalian collagen, type IX, alpha 2 gene (COL9A2) encodes the alpha-2 chain of type IX collagen and is located on horse chromosome 2p16-->p14 harbouring a quantitative trait locus for osteochondrosis. We isolated a bacterial artificial chromosome (BAC) clone containing the equine COL9A2 gene and determined the complete genomic sequence of this gene. Cloning and characterization of equine COL9A2 revealed that the equine gene consists of 32 exons spanning approximately 15 kb. The COL9A2 transcript encodes a single protein of 688 amino acids. Thirty two single nucleotide polymorphisms (SNPs) equally distributed in the gene were detected in a mutation scan of eight unrelated Hanoverian warmblood stallions, including one SNP that affects the amino acid sequence of COL9A2. Comparative analyses between horse, human, mouse and rat indicate that the chromosomal location of equine COL9A2 is in agreement with known chromosomal synteny relationships. The comparison of the gene structure and transcript revealed a high degree of conservation towards the other mammalian COL9A2 genes. We chose three informative SNPs for association and linkage disequilibrium tests in three to five paternal half-sib families of Hanoverian warmblood horses consisting of 44 to 75 genotyped animals. The test statistics did not reach the significance threshold of 5% and so we could not show an association of COL9A2 with equine osteochondrosis.

Amino Acid Sequence↗

PubMed1990

Characterization and expression of the unique calmodulin gene of Aspergillus nidulans.

Complete cDNA and genomic clones for the unique calmodulin (CaM) gene of the filamentous fungus Aspergillus nidulans have been isolated and characterized. The gene contains five introns, of which three are at unique positions relative to other CaM genes. The A. nidulans CaM gene is transcribed as a single, 0.85-kilobase mRNA species that encodes a predicted protein 84% identical (93% similar if conservative changes are considered) to vertebrate CaM. The complete cDNA was ligated into a lambda PL promoter-regulated bacterial expression vector to allow expression of A. nidulans CaM in Escherichia coli. The expressed protein was purified from bacterial lysates by phenyl-Sepharose chromatography and migrated as a single species on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of Ca2+, A. nidulans CaM exhibited a shift in apparent Mr identical to vertebrate CaM. The bacterially synthesized protein activated vertebrate CaM-dependent phosphodiesterase, CaM-dependent protein kinase II, and myosin light chain kinase with kinetics similar to vertebrate CaM. Isolated conidia (G0 spores) were germinated to induce synchronous cell cycle re-entry and the levels of CaM mRNA and protein determined. Both CaM and its mRNA were regulated during cell cycle re-entry. Calmodulin mRNA levels increased 20-fold as germlings progressed through the G1 phase, while CaM levels increased 2-fold prior to the initiation of DNA synthesis. Messenger RNA levels decreased during S-phase while protein levels increased an additional 2-fold, peaking at the onset of mitosis followed by a subsequent decrease as cells completed mitosis. Disruption of the CaM gene by site-specific homologous recombination was lethal, indicating that CaM is essential for cell cycle progression.

Amino Acid Sequence↗

PubMed2004

Rhodobacter capsulatus photoactive yellow protein: genetic context, spectral and kinetics characterization, and mutagenesis.

A gene for photoactive yellow protein (PYP) was previously cloned from Rhodobacter capsulatus (Rc), and we have now found it to be associated with genes for gas vesicle formation in the recently completed genome sequence. However, the PYP had not been characterized as a protein. We have now produced the recombinant RcPYP in Escherichia coli as a glutathione-S-transferase (GST) fusion protein, along with the biosynthetic enzymes, resulting in the formation of holo-RcPYP following cleavage of the GST tag. The absorption spectrum (with characteristic peaks at 435 and 375 nm) and the photocycle kinetics, initiated by a laser flash at 445 nm, are generally similar to those of Rhodobacter sphaeroides (RsPYP) but are significantly different from those of the prototypic PYP from Halorhodospira halophila (HhPYP), which has a single peak at 446 nm and has slower recovery. RcPYP also is photoactive when excited with near-ultraviolet laser light, but the end point is then above the preflash baseline. This suggests that some of the PYP chromophore is present in the cis-protonated conformation in the resting state. The excess 435 nm form in RcPYP, built up from repetitive 365 nm laser flashes, returns to the preflash baseline with an estimated half-life of 2 h, which is markedly slower than that for the same reaction in RsPYP. Met100 has been reported to facilitate cis-trans isomerization in HhPYP, yet both Rc and RsPYPs have Lys and Gly substitutions at positions 99 and 100 (using HhPYP numbering throughout) and have 100-fold faster recovery kinetics than does HhPYP. However, the G100M and K99Q mutations of RcPYP have virtually no effect on kinetics. Apparently, the RcPYP M100 is in a different conformation, as was recently found for the PYP domain of Rhodocista centenaria Ppr. The cumulative results show that the two Rhodobacter PYPs are clearly distinct from the other species of PYP that have been characterized. These properties also suggest a different functional role, that we postulate to be in regulation of gas vesicle genes, which are known to be light-regulated in other species.

Bacterial Proteins↗

PubMed1997

Characterization of the bovine C alpha gene.

The complete genomic sequence of a bovine C alpha gene is reported here. The genomic sequence was obtained from a C alpha phage clone that had been cloned from a genomic EMBL4 phage vector library. The C alpha sequence had previously been expressed as a chimeric antibody and identified as IgA using IgA-specific antibodies. Intron/exon boundaries were determined by comparison of the genomic sequence with an expressed bovine C alpha sequence obtained from spleen by reverse transcription-polymerase chain reaction (RT-PCR). Analysis of 50 Swedish bovine genomic DNA samples using genomic blots and five different restriction enzymes failed to detect evidence of polymorphism. However, PstI digests of Brown Swiss DNA showed a restriction fragment length polymorphism (RFLP), suggesting that at least two allelic variants of bovine IgA exist. Comparison of the deduced amino acid sequence of bovine IgA with sequences available for other species indicated that the highest homology was with that of swine, another artiodactyl. This was the highest homology observed for all mammalian IgA compared except for that between IgA1 and IgA2 in humans. Bovine IgA shares with rabbit IgA3 and IgA4, an additional N-linked glycosylation site at position 282. However, the collective data indicate that cattle are like swine and rodents and unlike rabbits in having a single locus of the gene encoding IgA of this species.

Amino Acid Sequence↗

PubMed2006

Using mRNAs lengths to accurately predict the alternatively spliced gene products in Caenorhabditis elegans.

MOTIVATION: Computational gene prediction methods are an important component of whole genome analyses. While ab initio gene finders have demonstrated major improvements in accuracy, the most reliable methods are evidence-based gene predictors. These algorithms can rely on several different sources of evidence including predictions from multiple ab initio gene finders, matches to known proteins, sequence conservation and partial cDNAs to predict the final product. Despite the success of these algorithms, prediction of complete gene structures, especially for alternatively spliced products, remains a difficult task. RESULTS: LOCUS (Length Optimized Characterization of Unknown Spliceforms) is a new evidence-based gene finding algorithm which integrates a length-constraint into a dynamic programming-based framework for prediction of gene products. On a Caenorhabditis elegans test set of alternatively spliced internal exons, its performance exceeds that of current ab initio gene finders and in most cases can accurately predict the correct form of all the alternative products. As the length information used by the algorithm can be obtained in a high-throughput fashion, we propose that integration of such information into a gene-prediction pipeline is feasible and doing so may improve our ability to fully characterize the complete set of mRNAs for a genome. AVAILABILITY: LOCUS is available from http://ural.wustl.edu/software.html

Algorithms↗

PubMed1993

Cloning and characterization of the calmodulin-encoding gene from Pneumocystis carinii.

Complete cDNA and genomic clones for the CaM gene encoding calmodulin (CaM) from Pneumocystis carinii have been isolated from rat and characterized. The nucleotide (nt) sequence contains an open reading frame interrupted by three introns, that encodes a protein of 152 amino acids. The predicted CaM protein of P. carinii shares a high degree of homology with other known CaM proteins.

Amino Acid Sequence↗