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PubMed1995

Deletion mapping of highly conserved transcribed sequence downstream from APRT locus.

To investigate the nature of DNA sequence rearrangements occurring in a highly malignant human colorectal carcinoma cell line (SW620) exhibiting a high level of chromosome instability, we characterized the molecular basis of deletions eliminating APRT. Deletions in SW620 resembled those in a variety of cell lines. They were joined at regions of little similarity through mono-, di-, or trinucleotide repeats. Breakpoint regions were rich in di- and trinucleotide repeats that might constitute pause sites for the replication complex. Deletions ranged in size from 1.8 to approximately 70 kb and were "directional" in that they eliminated sequences upstream of APRT but not downstream. Analysis of downstream sequences suggested that this pattern of deletion was due to the presence of another gene. Transcripts from these two genes converged but did not overlap. Given that this gene was not deleted in any hamster or human mutants, it appears essential for cell viability. This organization has important consequences for the pattern of mutation and repair of this region.

Adenine Phosphoribosyltransferase↗
PubMed1986

Characterization and structure of an endoglucanase gene cenA of Cellulomonas fimi.

Two BamHI fragments (0.8 and 5.2 kb) of Cellulomonas fimi containing an endoglucanase (Eng) gene (cenA) were individually cloned into the BamHI site of pBR322; they expressed carboxymethylcellulase activity in Escherichia coli. The nucleotide (nt) sequence of the cenA gene was determined by sequencing overlapping deletions. The cenA gene is 1350 bp long encoding a polypeptide of 449 amino acids (aa) and stop codon. The 0.8-kb BamHI component encodes the first 76 aa, whereas the 5.2-kb BamHI component encodes the rest of the Eng. The Eng lacking the N-terminal 76 aa retains its activity and antigenicity, and it forms an active fusion protein with the N-terminal portion of the TcR determinant. The C-terminal region of the Eng is crucial for activity and a deletion of as little as 12 aa from that end results in the loss of all Eng activity. The N-terminal 31 aa of the Eng constitute a leader peptide which appears to be functional in exporting the enzyme to the periplasm in E. coli.

Amino Acid Sequence↗
PubMed1999

FMRFamide-related neuropeptide gene family in Caenorhabditis elegans.

Neuropeptides are used as signaling molecules in the nervous system of most organisms, including mammals. The family of FMRFamide (Phe-Met-Arg-Phe-NH2)-like neuropeptides (FaRPs) all share an RFamide sequence at their C-termini and have been shown to have diverse functions in the central and peripheral nervous systems. In the nematode Caenorhabditis elegans, FMRFamide-like peptides (FaRPs) are expressed in at least 10% of the neurons, including motor, sensory, and interneurons that are involved in movement, feeding, defecation, and reproduction. Twenty-two genes, designated flp-1 through flp-22, encode FaRPs in C. elegans, although there are likely to be additional flp genes to be identified. Each flp gene encodes a different set of FaRPs, yielding a predicted total of 59 distinct FaRPs; a few of the genes may also encode non-FaRPs. Inactivation of some of the flp genes indicates that at least one flp gene has unique functions, while at least two flp genes appear to have overlapping functions with other flp genes. These results suggest that a complex family of FaRPs have varied roles through all stages of development and in adulthood in C. elegans.

Animals↗
PubMed1996

The sequence of the dictyostelium myo J heavy chain gene predicts a novel, dimeric, unconventional myosin with a heavy chain molecular mass of 258 kDa.

The complete sequence of the Dictyostelium myo J heavy chain gene has been determined from overlapping genomic clones. The gene spans approximately 7400 base pairs, is split by two small introns, and encodes a 2241-residue, 258-kDa heavy chain polypeptide that that is composed of an N-terminal 944-residue myosin head domain, a central 863-residue domain that is predicted to form an alpha helical coiled-coil containing six hinges, and a C-terminal 434-residue globular domain. The head domain is notable in that it contains a approximately 30 residue insert near the nucleotide binding pocket, and five potential calmodulin/myosin light chain binding sites at the head/tail junction. The existence within the Myo J tail domain of both an extensive coiled-coil structure and a large globular domain suggests that this myosin is dimeric and incapable of self-assembly into filaments. While these properties, as well as the overall predicted structure of the Myo J protein, are reminiscent of class V myosins, the sequence of the 434-residue globular tail piece of Myo J shows no similarity to that of either yeast or vertebrate myosins V. Consistent with this, phylogenetic analyses based on myosin head sequence comparisons do not classify Myo J as a type V myosin. These and other sequence comparisons indicate that Myo J and two as-yet-unclassified unconventional myosins from Arabidopsis represent members of the newest class within the myosin superfamily (class XI). Northern blots analyses suggest that Myo J may function predominantly in vegetative Dictyostelium cells. Finally, Southern blot analyses suggest that Dictyostelium possesses another myosin that is very closely related to Myo J.

Amino Acid Sequence↗
PubMed1997

Characterization of PKIgamma, a novel isoform of the protein kinase inhibitor of cAMP-dependent protein kinase.

Attempts to understand the physiological roles of the protein kinase inhibitor (PKI) proteins have been hampered by a lack of knowledge concerning the molecular heterogeneity of the PKI family. The PKIgamma cDNA sequence determined here predicted an open reading frame of 75 amino acids, showing 35% identity to PKIalpha and 30% identity to PKIbeta1. Residues important for the high affinity of PKIalpha and PKIbeta1 as well as nuclear export of the catalytic (C) subunit of cAMP-dependent protein kinase were found to be conserved in PKIgamma. Northern blot analysis showed that a 1.3-kilobase PKIgamma message is widely expressed, with highest levels in heart, skeletal muscle, and testis. RNase protection analysis revealed that in most tissues examined PKIgamma is expressed at levels equal to or higher than the other known PKI isoforms and that in several mouse-derived cell lines, PKIgamma is the predominant PKI message. Partial purification of PKI activities from mouse heart by DEAE ion exchange chromatography resolved two major inhibitory peaks, and isoform-specific polyclonal antibodies raised against recombinant PKIalpha and PKIgamma identified these inhibitory activities to be PKIalpha and PKIgamma. A comparison of inhibitory potencies of PKIalpha and PKIgamma expressed in Escherichia coli revealed that PKIgamma was a potent competitive inhibitor of Calpha phosphotransferase activity in vitro (Ki = 0.44 nM) but is 6-fold less potent than PKIalpha (Ki = 0.073 nM). Like PKIalpha, PKIgamma was capable of blocking the nuclear accumulation of Flag-tagged C subunit in transiently transfected mammalian cells. Finally, the murine PKIgamma gene was found to overlap the murine adenosine deaminase gene on mouse chromosome 2. These results demonstrate that PKIgamma is a novel, functional PKI isoform that accounts for the previously observed discrepancy between PKI activity and PKI mRNA levels in several mammalian tissues.

Amino Acid Sequence↗
PubMed2026

Integrative genomic and transcriptomic analysis of hypertension in a Taiwanese population.

OBJECTIVES: Hypertension is highly prevalent in Asian populations and represents a major cardiovascular risk factor. However, most genome-wide association studies (GWASs) and transcriptome-wide association studies (TWASs) have focused primarily on Caucasian cohorts. This study aimed to identify genetic loci and gene expression signatures associated with hypertension in an Asian population. METHODS: We analyzed 10 739 hypertensive patients and 49 668 controls from the Taiwan Biobank, testing 4 512 191 genome-wide single nucleotide polymorphisms (SNPs). Integrated GWAS, TWAS, and expression quantitative trait locus (eQTL) analyses were conducted to characterize genetic risk. Additionally, a polygenic risk score (PRS) was constructed using a split-sample design to evaluate genetic risk stratification. RESULTS: We identified 14 loci significantly associated with hypertension, including a novel locus at 5p13.1. eQTL analysis linked this locus to DAB2 expression in whole blood. TWAS detected 55 hypertension-associated genes, with 20 (36%) overlapping GWAS loci. Several novel genes outside GWAS loci, including FBXL15, KCNIP2, and CRIP3, were highly significant and implicated in vascular biology and hypertension mechanisms. PRS analysis effectively differentiated hypertension risk, with individuals in the top 10% showing a > 3.5-fold increased risk compared to the bottom 10%. CONCLUSIONS: Our findings provide new insights into the genetic and transcriptomic landscape of hypertension in Asians. The identification of novel loci and genes advances understanding of disease biology and may guide precision medicine approaches for risk prediction and therapeutic development.

Female↗
PubMed2025

Evaluating selection at intermediate scales within genes provides robust identification of genes under positive selection in M. tuberculosis clinical isolates.

Multiple studies have reported genes in the M. tuberculosis (Mtb) genome that are under diversifying selection, based on genetic variants among Mtb clinical isolates. These might reflect adaptions to selection pressures associated with modern clinical treatment of TB. Many, but not all, of these genes under selection are related to drug resistance. Most of these studies have evaluated selection at the gene-level. However, positive selection can be evaluated on different scales, including individual sites (codons) and local regions within an ORF. In this paper, we use GenomegaMap, a Bayesian method for calculating selection, to evaluate selection of genes in the Mtb genome at all three levels. We present evidence that the intermediate analysis (windows of codons) yields the most credible list of candidate genes under selection (excluding PPE and PE_PGRS genes, which are predicted less reliably due to frequent sequencing errors). A further advantage of this approach is that it identifies specific regions within proteins that are under selective pressure, which is useful for structural and functional interpretation. In an analysis of two separate collections of Mtb clinical isolates (from Moldova; and a globally-representative set), we observed 53 and 173 significant genes under selection, with 36% overlap. The lists of genes under selection include many drug-resistance genes, as well as other genes that have previously been reported to be under selection (resR, phoR). The specific regions under selection identified within drug-resistance genes are shown to correspond to protein structural features known to be involved in resistance, supporting accuracy of the method. Positive selection in several ESX-1-related genes was also observed, suggesting adaptation to immune pressure.

adaptation↗
PubMed1995

Characterization of the osmotically inducible gene osmE of Escherichia coli K-12.

osmE, an osmotically inducible gene of Escherichia coli, was physically mapped on the bacterial chromosome, cloned and sequenced. osmE appeared to encode a 12,021 Da protein of unknown function, with a lipoprotein-type signal sequence at the amino-terminus. The osmE reading frame was confirmed by sequencing the junction of an osmE-phoA gene fusion. osmE was demonstrated to be transcribed as a single cistron. A phi [osmEp-lac] operon fusion was constructed, and analysis of its expression demonstrated that osmE osmotic regulation probably occurs at the transcriptional level. The osmE promoter was identified by both S1 nuclease and primer extension mapping of the 5' end of the osmE mRNA, by deletion analysis and by identification of a point mutation reducing its activity. Sequence information sufficient for expression and osmotic regulation is present on a DNA fragment extending from positions -37 to +52 with respect to the osmE transcription start. Uninduced expression of the osmE-lac fusion was increased in the presence of mutations in the hns and himA genes. The osmE promoter overlaps a promoter for a gene transcribed in the opposite direction, efg. Transcription from the efg promoter is only weakly affected by osmotic pressure and is independent of the presence of an intact OsmE protein.

Amino Acid Sequence↗
PubMed1994

Genetic relationship between soxRS and mar loci in promoting multiple antibiotic resistance in Escherichia coli.

Multiple antibiotic resistance in Escherichia coli has typically been associated with mutations at the mar locus, located at 34 min on the E. coli chromosome. A new mutant, marC, isolated on the basis of a Mar phenotype but which maps to the soxRS (encoding the regulators of the superoxide stress response) locus located at 92 min, is described here. This mutant shares several features with a known constitutive allele of the soxRS gene, prompting the conclusion that it is a highly active allele of this gene. The marC mutation has thus been given the designation soxR201. This new mutant was used to examine the relationship between the mar and sox loci in promoting antibiotic resistance. The results of these studies indicate that full antibiotic resistance resulting from the soxR201 mutation is partially dependent on an intact mar locus and is associated with an increase in the steady-state level of mar-specific mRNA. In addition, paraquat treatment of wild-type cells is shown to increase the level of antibiotic resistance in a dose-dependent manner that requires an intact soxRS locus. Conversely, overexpression of MarA from a multicopy plasmid results in weak activation of a superoxide stress response target gene. These findings are consistent with a model in which the regulatory factors encoded by the marA and soxS genes control the expression of overlapping sets of target genes, with MarA preferentially acting on targets involved with antibiotic resistance and SoxS directed primarily towards components of the superoxide stress response. Furthermore, compounds frequently used to induce the superoxide stress response, including paraquat, menadione, and phenazine methosulfate, differ with respect to the amount of protection provided against them by the antibiotic resistance response.

Alleles↗
PubMed1985

Two functions of the E protein are key elements in the plasmid F replication control system.

By using a plasmid carrying a translational fusion between the E gene of the IncFI plasmid F and the lacZ gene, we located the operator of the autogenously regulated E gene to an inverted repeat overlapping the E-gene promoter and showing perfect homology to part of the sequence found in all the direct repeats of two regions exerting an inhibitory effect on F replication, incB and incC. Excess E protein provided in trans to an F plasmid increased the replication frequency of the F plasmid. This stimulatory effect was counteracted by increased dosages of incB or incC. A model is proposed for the replication control system of F in which the key elements are autoregulation of E-gene expression and titration of E protein by incB and incC.

Bacterial Proteins↗
PubMed1988

Organization and multiple regulation of histidine utilization genes in Pseudomonas putida.

The arrangement of the histidine utilization (hut) genes in Pseudomonas putida was established by examining the structure of a DNA segment that had been cloned into Escherichia coli via a cosmid vector. Southern blot analysis revealed that the restriction patterns of the hut genes cloned into E. coli and present in the P. putida genome were identical, indicating that no detectable DNA rearrangement took place during the cloning. Expression of the hut genes from a series of overlapping clones indicated the gene order to be hutG-hutI-hutH-hutU-hutC-hutF. The transcription directions of the different hut genes were determined by cloning the genes under control of the lambda pL promoter. This showed that hutF, encoding formiminoglutamate hydrolase, was transcribed in a direction opposite to that of the other genes. Inactivation of the cloned hut genes by Tn1000 insertion revealed that the hut genes were divided into three major transcriptional units (hutF, hutC [the repressor gene], and hut UHIG), but hutG may also be independently transcribed. When cloned individually with hutC on the same vector, hutF and hutU (which encodes urocanase) expression was induced by urocanate, indicating that these two genes each possess an operator-promoter element. Tn1000 insertions (in the cloned genes) or Tn5 insertions (in the P. putida genome) affecting the hutI or hutH gene only partially eliminated hutG expression. Furthermore, hutG, which specifies N-formylglutamate amidohydrolase, was regulated by the hutC product when the two genes were cloned on the same vector and expressed in E. coli. Therefore, hutG can be expressed independently from its own promoter, in keeping with earlier observations that N-formylglutamate amidohydrolase synthesis is not coordinated with that of urocanase and histidase and can be induced by N-formylglutamate or urocanate.

Cloning, Molecular↗
PubMed2001

Genetic organization of the region encoding regulation, biosynthesis, and transport of rhizobactin 1021, a siderophore produced by Sinorhizobium meliloti.

Eight genes have been identified that function in the regulation, biosynthesis, and transport of rhizobactin 1021, a hydroxamate siderophore produced under iron stress by Sinorhizobium meliloti. The genes were sequenced, and transposon insertion mutants were constructed for phenotypic analysis. Six of the genes, named rhbABCDEF, function in the biosynthesis of the siderophore and were shown to constitute an operon that is repressed under iron-replete conditions. Another gene in the cluster, named rhtA, encodes the outer membrane receptor protein for rhizobactin 1021. It was shown to be regulated by iron and to encode a product having 61% similarity to IutA, the outer membrane receptor for aerobactin. Transcription of both the rhbABCDEF operon and the rhtA gene was found to be positively regulated by the product of the eighth gene in the cluster, named rhrA, which has characteristics of an AraC-type transcriptional activator. The six genes in the rhbABCDEF operon have interesting gene junctions with short base overlaps existing between the genes. Similarities between the protein products of the biosynthesis genes and other proteins suggest that rhizobactin 1021 is synthesized by the formation of a novel siderophore precursor, 1,3-diaminopropane, which is then modified and attached to citrate in steps resembling those of the aerobactin biosynthetic pathway. The cluster of genes is located on the pSyma megaplasmid of S. meliloti 2011. Reverse transcription-PCR with RNA isolated from mature alfalfa nodules yielded no products for rhbF or rhtA at a time when the nifH gene was strongly expressed, indicating that siderophore biosynthesis and transport genes are not strongly expressed when nitrogenase is being formed in root nodules. Mutants having transposon insertions in the biosynthesis or transport genes induced effective nitrogen-fixing nodules on alfalfa plants.

Amino Acid Sequence↗
PubMed2005

[Construction of recombinant adeno-associated virus vectors carrying double gene of antisense multidrug resistance-associated protein and antisense multidrug resistance].

OBJECTIVE: To construct a recombinant adeno-associated virus vectors carrying double gene of antisense multidrug resistance-associated protein (MRP) and antisense multidrug resistance (MDR1) for use in studying the gene therapy to reverse the multidrug resistance (MDR) in hepatocellular carcinoma (HCC). METHODS: The 500 bp fragment (mrp) of MRP cDNA 5' region and the 600 bp fragment (mdr1) of MDR1 cDNA 5' region were amplified through polymerase chain reaction (PCR), and then they were linked to a combined gene fragment (mrp+mdrl) by overlapping technique. The combined gene fragment(mrp+mdrl) was cloned reversely into the multiple cloning site (MCS) of the expression plasmid pAAV-IRES-hrGFP in AAV Helper-Free System to construct the recombinant expression plasmid pAAV-IRES-hrGFP-(mrp + mdr1)AS. The packaging cell line (HEK 293 cell) was co-transfected with the pAAV-IRES-hrGFP-(mrp+mdr1)AS together with the control plasmid pAAV-RC and pHelper in AAV Helper-Free System by means of lipofectamine. The recombinant adeno-associated virus vector : rAAV2-(mrp+mdr1)AS carrying the double gene of antisense multidrug resistance-associated protein (MRP)and antisense multidrug resistance (MDR1) was packaged. Then the viral titer was checked by GFP. RESULTS: The recombinant adeno-associated virus vector : rAAV2-(mrp + mdr1)AS carrying antisense MRP and antisense MDR1 was constructed successfully, the strong green fluorescence was observed in HEK 293 cells under a fluorescence microscope. The viral titer was 2.5 X 10(6) efu/ml. CONCLUSION: The rAAV2-(mrp+mdr1)AS thus constructed could introduce the antisense MRP and antisense MDR1 into the human drug-resistant hepatocellular cell line effectively, which might provide a sound basis for the mechanisms and reversal methods of the multidrug resistance in HCC.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Expression of rTS correlates with altered growth regulation of thymidylate synthase.

The recently discovered rTS gene is convergent with and overlaps the thymidylate synthase gene in the antisense orientation. K562 B1A and KB 1BT are two methotrexate-resistant cell lines that have amplified genes for dihydrofolate reductase. K562 B1A cells have increased levels of rTS mRNA and protein compared with their parental K562 cells, whereas KB 1BT cells show unaltered rTS expression compared with their parental (KB) cells. Altered levels and loss of growth-regulation of in situ thymidylate synthase activity were found in methotrexate-resistant K562 B1A cells but not in K562, KB, and KB 1BT cells. These data point to a link between rTS and the regulation of TS activity.

Blotting, Northern↗
PubMed2004

Identifying spatially similar gene expression patterns in early stage fruit fly embryo images: binary feature versus invariant moment digital representations.

BACKGROUND: Modern developmental biology relies heavily on the analysis of embryonic gene expression patterns. Investigators manually inspect hundreds or thousands of expression patterns to identify those that are spatially similar and to ultimately infer potential gene interactions. However, the rapid accumulation of gene expression pattern data over the last two decades, facilitated by high-throughput techniques, has produced a need for the development of efficient approaches for direct comparison of images, rather than their textual descriptions, to identify spatially similar expression patterns. RESULTS: The effectiveness of the Binary Feature Vector (BFV) and Invariant Moment Vector (IMV) based digital representations of the gene expression patterns in finding biologically meaningful patterns was compared for a small (226 images) and a large (1819 images) dataset. For each dataset, an ordered list of images, with respect to a query image, was generated to identify overlapping and similar gene expression patterns, in a manner comparable to what a developmental biologist might do. The results showed that the BFV representation consistently outperforms the IMV representation in finding biologically meaningful matches when spatial overlap of the gene expression pattern and the genes involved are considered. Furthermore, we explored the value of conducting image-content based searches in a dataset where individual expression components (or domains) of multi-domain expression patterns were also included separately. We found that this technique improves performance of both IMV and BFV based searches. CONCLUSIONS: We conclude that the BFV representation consistently produces a more extensive and better list of biologically useful patterns than the IMV representation. The high quality of results obtained scales well as the search database becomes larger, which encourages efforts to build automated image query and retrieval systems for spatial gene expression patterns.

Algorithms↗
PubMed1995

Overlapping pathways mediate the opposing actions of tumor necrosis factor-alpha and transforming growth factor-beta on alpha 2(I) collagen gene transcription.

Transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) are multifunctional peptides intimately involved in the process of extracellular matrix remodeling. We recently showed that TGF-beta stimulates the human alpha 2(I) collagen gene by increasing the affinity of an Sp1-containing transcriptional complex bound to an upstream sequence termed the TbRE (Inagaki, Y., Truter, S. and Ramirez, F. (1994) J. Biol. Chem. 269, 14828-14834). Here, we report that the TbRE-bound complex also mediates the inhibitory signal of TNF-alpha. Nuclear proteins from cells treated with TNF-alpha bind to the TbRE sequence substantially more strongly than those from untreated cells. Additionally, TNF-alpha increases binding of a second protein complex that recognizes the negatively cis-acting element located immediately next to the TbRE. Thus, we postulate that TNF-alpha counteracts the TGF-beta-elicited stimulation of collagen gene expression through overlapping nuclear signaling pathways. One modifies the TGF-beta-targeted transcriptional complex, probably by reducing its stimulatory effect on collagen transcription. The other acts on the binding of the adjacent factor, presumably by increasing its effectiveness in repressing the activity of the collagen promoter. The convergence of the TGF-beta and TNF-alpha pathways on the same sequence of the alpha 2(I) collagen promoter is yet another example of combinatorial gene regulation achieved through composite response elements.

Base Sequence↗
PubMed1992

Gene junction sequences of bovine respiratory syncytial virus.

The nucleotide sequences of seven gene junctions (N-P, P-M, M-SH, SH-G, G-F, F-M2 and M2-L) of bovine respiratory syncytial virus (BRSV) strain A51908 were determined by dideoxynucleotide sequencing of cDNAs from polytranscript mRNAs and from genomic RNA. By comparison with the consensus sequences derived from human respiratory syncytial virus (HRSV) mRNAs, gene-start and gene-end sequences were found in all BRSV mRNAs. There was a perfect match between the BRSV and HRSV in all gene-start sequences, except for the sequence of the SH gene which contained one nucleotide difference compared to HRSV A2; and the gene-start sequence of the L gene, which was one nucleotide shorter than the corresponding sequence of HRSV. Analysis of the intergenic regions showed a high degree of divergence in the nucleotide sequence between BRSV and HRSV. However, the length of the nucleotides in the intergenic sequences was similar for a given gene junction. As in the case of HRSV, the M2 and L genes of BRSV overlap by 68 nucleotides, suggesting a similar transcription attenuation mechanism. The sequences of the overlap, corresponding to the 3' end of the L gene, were almost identical between BRSV and HRSV.

Animals↗
PubMed2006

Gene expression-based chemical genomics identifies rapamycin as a modulator of MCL1 and glucocorticoid resistance.

Drug resistance remains a major obstacle to successful cancer treatment. A database of drug-associated gene expression profiles was screened for molecules whose profile overlapped with a gene expression signature of glucocorticoid (GC) sensitivity/resistance in acute lymphoblastic leukemia (ALL) cells. The screen indicated that the mTOR inhibitor rapamycin profile matched the signature of GC sensitivity. We tested the hypothesis that rapamycin would induce GC sensitivity in lymphoid malignancy cells and found that it sensitized to GC-induced apoptosis via modulation of antiapoptotic MCL1. These data indicate that MCL1 is an important regulator of GC-induced apoptosis and that the combination of rapamycin and glucocorticoids has potential utility in lymphoid malignancies. Furthermore, this approach represents a strategy for identification of promising combination therapies for cancer.

Animals↗