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At least 883 records · Page 49Linked to original sources
PubMed2003

Genomic organization and characterization of the mouse ELYS gene.

Differentiation of hematopoietic stem cells into blood cells is controlled by several transcription factors. Recently, we identified a putative transcription factor, ELYS (for embryonic large molecule derived from yolk sac), using a subtraction strategy. During mouse embryogenesis, ELYS transcripts were predominantly expressed in hematopoietic tissues, such as the yolk sac, aorta-gonad-mesonephros (AGM), and liver. Here, we report the cloning and characterization of the mouse ELYS gene. The ELYS gene spanned approximately 60kb encoding 36 exons, and was assigned between D1Mit315 and D1Mit458 markers in chromosome 1. The transcription initiation site was identified as the G residue located 670bp upstream of the translation start codon. A region downstream of the transcriptional start site contributed to high promoter activity. This region contained potential DNA elements for transcription factors such as GATA-1, -2, -3, heat shock factor (HSF) 2, and NF-kappaB, which are known to play important roles in hematopoietic events.

Animals↗
PubMed2005

Genomic organization, promoter characterization and roles of Sp1 and AP-2 in the basal transcription of mouse PDIP1 gene.

The mouse polymerase delta-interacting protein 1 gene, PDIP1, is mapped to chromosome 7F3 region, spans approximately 16.7kb, and is organized into six exons. The transcription start site (TSS) was determined to be G, corresponding to position of 162-bp upstream of the translation start codon. The promoter region was found to lack TATA box or CCAAT box, instead, a CpG island was detected surrounding TSS. The region from -162 to +114 is required for basal transcriptional regulation of mouse PDIP1 gene, contains two AP-2 and two Sp1 binding sites. The Sp1 site upstream of TSS activates, while the other Sp1 site and two AP-2 sites suppress the transcription activity of mouse PDIP1 gene.

Animals↗
PubMedDate not supplied

Genomic identification and characterization of avian adenoviruses associated with inclusion body hepatitis.

Four pathogenic avian adenovirus isolates associated with inclusion body hepatitis and mortality in commercial broiler chicks and chickens were characterized and identified. These group I avian adenovirus isolates were classified as group E (serotypes 6, 7, 8, and 9) avian adenoviruses on the basis of the restriction enzyme patterns of their viral DNA. Isolate 3718 was neutralized by a serotype 6 reference avian adenovirus antiserum and isolates 8193, 8380, and 8565 were all neutralized by a serotype 8 reference avian adenovirus antiserum by virus neutralization assays. Infectivity and virulence such as mortality, hemorrhages, enlarged green livers with intranuclear inclusion bodies, stunting, intestinal sloughing, and poor feathering were observed in specific-pathogen-free chicken embryos and were identical for all four isolates when embryos were inoculated via the yolk sac and/or chorioallantoic membrane. Complete mortality was observed within 72 hr postinoculation in specific-pathogen-free (SPF) chickens inoculated intramuscularly for all four avian adenovirus isolates.

Adenoviridae↗
PubMed2004

Mutated E-cadherin: genomic and functional characterization in thyroid cells from the KAT family.

Members of a family of thyroid cell lines (KAT) were analyzed because they expressed a higher molecular weight (135 kd) form of E-cadherin at their surface. We found that this aberrant E-cadherin is the result of a point mutation in the exon 9 donor splice site causing a skipping of exon 9 with consequent deletion of the corresponding aminoacids on E-cadherin protein. As a spin-off, we report that the various members of the KAT family share this mutation as well as the genetic background. Furthermore we found that this mutated protein leads to disturbed cell-cell adhesion although E-cadherin is still able to mediate the formation of the cadherin/ catenin complex. We also demonstrate the presence of another cell-cell adhesion complex, formed by Pcadherin and the catenins. The latter is also not able to mediate cell-cell adhesion. Although these cells lack cell-cell adhesion they are not invasive without exogenous stimulus.

Base Sequence↗
PubMed1993

Mapping the sheep genome: production of characterized sheep x hamster cell hybrids.

Specific chromosomes of sheep have been selectively "captured" in hamster x sheep cell hybrids produced by fusing different Chinese hamster auxotrophs with lymphocytes from sheep carrying normal or Robertsonian translocation chromosomes. A minipanel has been established comprising sheep chromosomes 1; 3; t1 = rob(6;24); t2 = rob(9;10); and X in a predominantly monochromosomal state. Using this targeted cell fusion approach the nutritional markers, uridine monophosphate synthetase (UMPS), mapped onto human chromosome 3 (HSA3), phosphoribosylglycinamide synthetase, and phosphoribosylaminoimidazole synthetase (GART) (HSA21), are reported to be located on sheep chromosome 1q; and phosphoribosyl pyrophosphate amidotransferase (PPAT) (HSA4) has been assigned to sheep chromosome 6. By isozyme analysis and Southern hybridization, transferrin (TF) and superoxide dismutase 1 (SOD1), both in the bovine syntenic group U10, were assigned to sheep chromosome 1q; adenylate kinase 1 (AK1), in the bovine syntenic group U16, to sheep chromosome 3p; lactate dehydrogenase B (LDHB) and phenylalanine hydroxylase (PAH) on bovine chromosome 5 (BTA5) to sheep chromosome 3q; phosphoglucomutase 2 (PGM2) (bovine syntenic group 15) to sheep chromosome 6 (t1q); avian myelocytomatosis viral oncogene homolog (MYC) and Moloney murine sarcoma viral oncogene homolog (MOS) (BTA 14) to sheep 9 (t2q); and glutathione reductase (GSR) (bovine syntenic group U14) to sheep 24 (t1p).

Animals↗
PubMed2006

Genomic organization, promoter characterization and expression profiles of an antiviral gene PmAV from the shrimp Penaeus monodon.

Diseases caused by viruses are the greatest challenge to worldwide shrimp aquaculture. PmAV is the first antiviral gene identified from the shrimp and its mRNA level is up-regulated in response to viral infection (Luo et al., 2003). The transcriptional regulation of PmAV is critical since the expression of this gene is regulated in response to viral stimuli. In this study, we show that the Penaeus monodon PmAV gene contains four exons, separated by three introns. The 5'-flanking sequence of PmAV gene consists of a typical TATA box, several potential regulatory elements, and a compound microsatellite repeat as well. Interestingly, a TATA box and several regulatory elements also exist in the first intron (intron 1). Both intron 1 and the microsatellite are involved in transcriptional regulation of the PmAV gene. PmAV promoter activity depends on the existence of intron 1 while the microsatellite in 5'-flanking sequence acts as a negative regulative element. In addition, using Real-time PCR, we found that PmAV was highly expressed in the hepatopancreas and was up-regulated from the second day post-infection with the increase of viral load. These results extend our previous findings and provide insights into the molecular regulation of PmAV gene expression, which will be helpful for shrimp viral disease control.

Animals↗
PubMed1999

Genomic cloning and characterization of the rat glutathione S-transferase-A3-subunit gene.

The rat glutathione S-transferase-A3-subunit (GSTA3) gene is a member of the class Alpha GSTs, which we have previously reported to be overexpressed in anti-cancer-drug-resistant cells. In this study, we report the isolation and characterization of the entire rat GSTA3 (rGST Yc1) subunit gene. The rat GSTA3 subunit gene is approximately 15 kb in length and consists of seven exons interrupted by introns of different lengths. Exon 1, with a length of 219 bp, contains only the 5'-untranslated region of the gene. Each exon-intron splicing junction exhibited the consensus sequence for a mammalian splice site. The transcription start site and exon 1 of rat GSTA3 were characterized by a combination of primer extension and rapid amplification of the cDNA ends. Position +1 was identified 219 bp upstream of the first exon-intron splicing junction. The proximal promoter region of the rat GSTA3 subunit gene does not contain typical TATA or CAAT boxes. A computer-based search for potential transcription-factor binding sites revealed the existence of a number of motifs such as anti-oxidant-responsive element, ras-response element, activator protein-1, nuclear factor-kappaB, cAMP-response-element-binding protein, Barbie box and E box. The functional activity of the regulatory region of the rat GSTA3 subunit gene was shown by its ability to drive the expression of a chloramphenicol acetyltransferase reporter gene in rat mammary carcinoma cells, and its activity was greater in melphalan-resistant cells known to have transcriptional activation of this gene by previous studies. The structure of the gene, with a large intron upstream of the translation-initiation site, may explain why the isolation of this promoter has been so elusive. This information will provide the opportunity to examine the involvement of the rat GSTA3 subunit gene in drug resistance and carcinogenesis.

5' Untranslated Regions↗
PubMed2000

Cloning, genomic organization, and characterization of a human cholinephosphotransferase.

A cholinephosphotransferase activity catalyzes the final step in the de novo synthesis of phosphatidylcholine via the transfer of a phosphocholine moiety from CDP choline to diacylglycerol. Ethanolaminephosphotransferase activity catalyzes a similar reaction substituting CDP ethanolamine as the phosphobase donor. We report the identification and cloning of a human cDNA (human cholinephosphotransferase (hCPT1)) that codes for a cholinephosphotransferase-specific enzyme. This was demonstrated using in vitro enzyme assays and in vivo measurement of the reconstitution of the phosphatidylcholine and phosphatidylethanolamine biosynthetic pathways in yeast cells devoid of their own endogenous cholinephosphotransferase and ethanolaminephosphotransferase activities. This contrasted with our previously cloned human choline/ethanolaminephosphotransferase cDNA that was demonstrated to code for a dual specificity choline/ethanolaminephosphotransferase. The hCPT1 and human choline/ethanolaminephosphotransferase (hCEPT1) predicted amino acid sequences possessed 60% overall identity and had only one variation in the amino acid residues within the CDP-alcohol phosphotransferase catalytic motif. In vitro assessment of hCPT1 and hCEPT1 derived cholinephosphotransferase activities also revealed differences in diradylglycerol specificities including their capacity to synthesize platelet-activating factor and platelet-activating factor precursor. Expression of the hCPT1 mRNA varied greater than 100-fold between tissues and was most abundant in testis followed by colon, small intestine, heart, prostate, and spleen. This was in marked contrast to the hCEPT1 mRNA, which has been found in similar abundance in all tissues tested to date. Both the hCPT1 and hCEPT1 enzymes were able to reconstitute the synthesis of PC in yeast to levels provided by the endogenous yeast cholinephosphotransferase; however, only hCEPT1-derived activity was able to complement the yeast CPT1 gene in its interaction with SEC14 and affect cell growth.

Amino Acid Sequence↗
PubMed2002

Mouse testin: complementary DNA cloning, genomic organization, and characterization of its proximal promoter region.

Testin is a secretory protein that was initially identified from rat Sertoli cell-enriched cultures and has been suggested to be a sensitive marker to monitor the integrity of Sertoli-germ cell junctions. However, the expression of the testin gene in other species and the molecular mechanisms that govern its transcription are unknown. To address these issues, we cloned and characterized the mouse testin gene. A full-length mouse testin cDNA encoding a polypeptide of 333 amino acid residues was isolated by library screening. Sequence analysis revealed that mouse testin shares 90.1%, 58.9%, 62.2%, and 64.6% identity with rat testin and cathepsin L of mouse, rat, and human, respectively, at the amino acid level. Reverse transcription-polymerase chain reaction and Southern blot analysis demonstrated that mouse testin transcripts were predominantly expressed in the gonads. The mouse testin gene spans over 21 kilobases (kb) and contains eight exons interrupted by seven introns. Primer extension analysis and 5' rapid amplification of cDNA ends identified a major transcription start site located 134 base pairs upstream from the translation initiation codon. Analysis of a 2.3-kb mouse testin 5'-flanking region revealed that it lacked TATA and CAAT boxes, and the region was not GC rich. By the use of deletion analysis, in vitro DNase I footprinting, and site-directed mutagenesis, we identified within the proximal promoter region three closely spaced putative binding sites for GATA, sex-determining factor, and steroidogenic factor 1 that are important for testin gene transcription in mouse Sertoli (MSC-1) cells. These cis-acting elements are also present in the conserved Mullerian-inhibiting substance (MIS) proximal promoters, raising a possibility that the transcriptions of testin and MIS genes are controlled by similar mechanisms.

Amino Acid Sequence↗
PubMed1996

Cloning, mapping and characterization of a genomic copy of the Lipomyces kononenkoae alpha-amylase-encoding gene (LKA1).

The expression in Saccharomyces cerevisiae and Schizosaccharomyces pombe of a cDNA copy of the Lipomyces kononenkoae IGC4052B alpha-amylase gene (LKA1), linked to the phosphoglycerate kinase gene (PGK1) promoter, resulted in the extracellular production of biologically active alpha-amylase (LKA1). However, transformation of S. cerevisiae and Schiz. pombe with a cosmid clone containing the complete genomic copy of LKA1, expressed from its native promoter, did not result in secretion of active alpha-amylase by any of the transformants. When the cDNA copy of LKA1 was expressed in S. cerevisiae under control of the wild-type L, kononenkoae promoter, biologically active alpha-amylase was secreted into the culture medium, indicating the recognition of the LKA1 promoter in S. cerevisiae. Sequence analysis of the GC-rich LKA1 promoter revealed canonical sequences that are homologous to the TATAAA, CAAT and CCAAT boxes and GCN4-binding sites that are present in several promoter sequences of S. cerevisiae. Primer extension analysis of LKA1 transcripts in L. kononenkoae indicated major initiation sites at nucleotides -64 and -65. S. cerevisiae and Schiz. pombe cells transformed with a plasmid containing the open reading frame of the genomic copy of LKA1, linked to the PGK1 promoter, did not produce alpha-amylase. Polymerase chain reaction mapping and sequence analysis revealed the presence of a 61-bp intron in the genomic copy of LKA1 that impaired synthesis of biologically active alpha-amylase in S. cerevisiae and Schiz. pombe. This intron contains donor, acceptor and branch sequences that correlate with the consensus sequences identified in the introns of split genes from Schiz. pombe and mammals. Pulsed-field gradient gel electrophoresis resolved at least eight chromosomal DNAs for L. kononenkoae IGC4052B and chromoblot analysis indicated that LKA1 is located on the second smallest chromosome, designated chromosome II.

Ascomycota↗
PubMed2000

A 38 kb segment containing the cdc2 gene from the left arm of fission yeast chromosome II: sequence analysis and characterization of the genomic DNA and cDNAs encoded on the segment.

A genomic 38 kbp segment on the c1750 cosmid clone containing the cdc2 gene, located in the left arm of chromosome II from Schizosaccharomyces pombe, was sequenced. The segment was found to have five previously known genes, pht1, cdc2, his3, act1 and mei4. Among 11 coding sequences (CDSs) predicted by the gene finding software INTRON.PLOT., four CDSs, pi007, pi010, pi014 and pi016, had considerable similarity to 40S ribosomal protein, glycosyltransferase, cdc2-related protein kinase and alpha-1, 2-mannosyltransferase, respectively. Another unusually huge open reading frame (ORF) (pi011), consisting of 2233 amino acids, existed, having significant homology to alpha-amylase, granule-bound glycogen synthase and the Sz. pombe YS 1110 clone product at the N-terminal, middle and C-terminal regions, respectively. All the predicted 11 CDSs were experimentally analysed by RACE PCR. The sequencing of the RACE products revealed that there were two small overlaps at the 3' untranslated regions (UTRs) between pi004 and pi005 (17 bp) and between pi007 and pi008 (2 bp). The distances between 5' end of the 5'UTR and the putative translation initiation codon varied from 10 to 302 nucleotides (nt) among the nine CDSs successfully analysed by 5'-RACE. The expression level of each CDS on this clone was determined. Among the 16 genes on this clone, the previously determined genes, pht1, cdc2, his3 and act1, were found to be most highly expressed. Finally, cDNAs of all the newly identified genes were detected by RACE, proving the actual expression of these genes. The nucleotide sequence has been submitted to the EMBL database under Accession No. AB004534.

Base Sequence↗
PubMed1996

Loss of the chromosomal region 5q11-q31 in the myeloid cell line HL-60: characterization by comparative genomic hybridization and fluorescence in situ hybridization.

Comparative genomic hybridization was used to identify the regions of genomic gain and loss in the myeloid cell line HL-60. These included amplification at 8q24 corresponding to previous reports of overrepresentation of the MYC gene; loss of material from the short arms of chromosomes 9 (9p21-p23), 10, and 17; loss of the chromosome regions 9q32-qter and 14q11-q24; and an extra copy of chromosome 18. Additionally, deletion of the 5q11-q31 region was noted and was associated with translocation of chromosome 5 material to chromosomes 16 and a dic(5;17)(q11;p11) chromosome (previously described as mar 3). Loss of chromosome 5 material in myeloid malignancies, including the M2 subtype from which HL-60 was derived, is usually associated with interstitial deletions of the long arm, including the critical 5q31 region, resulting in a 5q- chromosome. The HL-60 cell line may be a useful model to investigate the role of potential tumour suppressor genes associated with loss of 5q material in myeloid leukaemias.

Chromosome Deletion↗
PubMed1999

Characterization of the genomic structure of the murine Spam1 gene and its promoter: evidence for transcriptional regulation by a cAMP-responsive element.

The Sperm Adhesion Molecule 1 (SPAM1), also known as PH-20, is a sperm membrane-bound protein that has been shown to have bifunctional roles in fertilization. It is encoded by a gene that is widely conserved in mammalian species, underscoring its importance in the fertilization process. Here we determined the genomic structure of the murine homologue, Spam1, using PCR analysis, and studied its transcriptional regulation. The gene covers approximately 10.5 kb of genomic DNA, is encoded by four exons, and the splice site consensus sequence is maintained in all intron-exon junctions, similar to that reported for the human homologue. With primer extension analysis, two transcription initiation sites were detected. One was assigned to the residue C and the other (a minor site) to the residue G, at positions 1 and -56, respectively. These are at 313 and 369 nucleotides upstream of the translation initiation codon, ATG. In about 770 bp upstream region of Spam1 that has been cloned and sequenced, multiple transcription factor binding sites including a CRE (cAMP-responsive element) were found. We specifically studied the function of the eight nucleotide CRE sequence (TGATGTCA) at -57 (or -62 depending on the strain of mice) of the promoter region. It can bind to the transcription factor CREM (cAMP-responsive element modulator) in gel mobility shift assays using mouse testis nuclear extract, and the binding can be inhibited by a 28 bp oligonucleotide containing the CRE sequence. Similar binding and inhibition assays using rat nuclear extract suggest the existence of a rat CRE sequence and the involvement of CREM in rat Spam1 expression. In vitro transcription assays suggest that CRE is necessary for the transcriptional activity of the murine promoter, and Northern analysis shows that Spam1 transcripts are absent in CREM-knockout mice. The results strongly suggest that the murine Spam1 expression is under the control of CREM, and that this transcriptional regulator for Spam1 might be conserved in other mammals, at least in the rat.

Animals↗
PubMed2000

Structure of the MLT gene and molecular characterization of the genomic breakpoint junctions in the t(11;18)(q21;q21) of marginal zone B-cell lymphomas of MALT type.

The t(11;18)(q21;q21) between the inhibitor of apoptosis API2 and the MLT gene is a distinct feature of marginal zone B-cell lymphomas of MALT-type. Hitherto the chimeric API2-MLT transcripts are all "in-frame" and predominantly fuse exon 7 of API2 to different MLT exons. Recurrent chromosomal translocations are common in lymphoid neoplasms and might represent by-products of the rearrangement processes generating antigen receptor diversity. The genomic structure of the MLT gene was determined to facilitate amplification of the genomic breakpoint junctions from 5 MALT-type lymphomas with t(11;18). Their sequence analysis showed scattering of the chromosome 11 breakpoints in intron 7 of API2 whereas rearrangements in MLT occurred in intron 2, 4, 7, or 8, respectively. Sequences around the junctions did not display recognition signal sequences mediating lymphocytic V(D)J recombination or other sequence motifs associated with recombination. The breakpoints occurred in a copy of an AluSx repeat in three cases, but interchromosomal Alu-mediated homologous recombination could be ruled out as the repeat resided only on one of the participating chromosomes. The t(11;18) was associated with a deletion in 4 out of 5 cases, ranging in size from 53 bp up to more than 200 kb. These deletions were observed on one or sometimes both derivative chromosomes that might indicate the susceptibility of these regions for breakage. Our data suggest that the API2-MLT fusion might result from a non-homologous end joining event after multiple double-strand breaks. The clustering of breaks in intron 7 of API2 and the consistent "in frame" API2-MLT fusions could therefore reflect certain functional constraints crucial for clonal outgrowth.

Apoptosis↗
PubMed2003

Quantitative characterization of specific genomic promoters using agarose gel electrophoresis.

Over the past decade a large number of studies have focused attention on the role of nucleosomes as negative and positive regulators of specific nuclear functions. Due to the lack of an analytical method to determine the higher order conformation of the nucleosomal arrays that encompass specific genetic loci (e.g., promoters, enhancers), research emphasis has mostly been centered on chromatin remodeling and histone posttranslational modifications. We have recently developed an agarose gel electrophoresis method that permits us to analyze the higher order structure of specific in vivo assembled chromatin fragments. After calibration using a well-defined in vitro system, we have been able to experimentally determine the size, shape, and conformational flexibility of the Mouse Mammary Tumor Virus long-terminal repeat promoter region in its repressed and activated states. These studies pave the way for widespread analyses of the higher order structure of specific, functionally important chromosomal loci, and in so doing enhance our understanding of the roles that the higher order structure of chromatin play in genome regulations.

Animals↗
PubMed1982

Characterization of viral genomes in the liver and serum of chimpanzee long-term hepatitis B virus carriers: a possible role for supercoiled HBV-DNA in persistent HBV infection.

In chimpanzee hepatitis B virus (HBV) carriers, the molecular mechanism for viral persistence has been examined by analyzing the properties of viral DNA molecules in liver and serum. Two extrachromosomal HBV-DNA molecules migrating on Southern blots at 4.0 kb and 2.3 kb were observed in chimpanzee liver DNA. There was no evidence for integration of HBV sequences into the host genome. The HBV-DNA molecule which migrated at 4.0 kb position represents a full-length "nicked," relaxed circular form, and the DNA molecules migrating at 2.3 kb position represents a supercoiled form of the HBV genome. Evidence for supercoiled HBV-DNA in serum was obtained by production of the relaxed circular intermediate upon digestion of Dane particle DNA with specific nucleases S1 and Bal 31. A possible role of these two extrachromosomal HBV-DNA molecules in the biology of hepatitis B virus infection and the mechanism for viral persistence are discussed.

Animals↗
PubMed1989

Characterization of two genome types of adenovirus type 31 isolated in Stockholm during 1987.

The authors isolated during 1987 seven adenovirus type 31 (Ad31) within a 9-month period. The isolates were obtained from urine, throat, and feces, implying a systemic spread of the infection. Most patients displayed gastrointestinal symptoms, but some had respiratory symptoms and fever. All of the strains differed by restriction endonuclease analysis from the prototype strain (1315) by an additional Bgl II restriction site. Ad31 isolates 1-6 could be divided into two groups by the enzymes Bam HI, Msp I, and Xho I. Each enzyme gave rise to the same group distribution: isolates 1-3 and 4-6, respectively. Digestion with Bst EII, Hind III, Kpn I, and Sma I resulted in identical patterns for isolates 1-6. Isolate 7, however, demonstrated a DNA deletion of approximately 0.8 kbp, but it was otherwise identical to isolates 4-6. In conclusion, two separate genome types of Ad31 were isolated, one of which included a DNA deletion mutant. The increased isolation rate may reflect an epidemiological situation, as the same isolation procedure had been used both before and after this period.

Adenovirus Infections, Human↗