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PubMed1997

Characterization of the murine cyclin D2 gene: exon/intron organization and promoter activity.

Cyclin D2 is normally expressed in G1 and promotes progression through G1 of the cell cycle. From a murine genomic library constructed with spleen DNA, two overlapping genomic clones of cyclin D2 were isolated. These clones contain most of the exon of cyclin D2 except exon 5. Characterization of these clones revealed that murine cyclin D2 mRNA spans over 18 kb and 5 exons ranging from 149 to approximately 462 bp in length, and suggested that exon 5 may be at least >5 kb downstream from exon 4. Primer extension analysis of cyclin D2 mRNA isolated from murine activated T cells detected 5 putative sites of transcription initiation. These are located at - 499, - 417, - 391, - 373, and - 349 relative to the translation start site, which is given as + 1. No consensus sequence for TATA box existed at an appropriate position within the promotor region. Instead, several putative transcriptional factor binding sites for C/EBP, PEA3, AP2, NF-Y, Sp1, c-Myc, GATA-1, AP1, v-Myb, and CREB were detected. The 5'-flanking region of the cyclin D2 gene up to nucleotide - 945 shared about 61% sequence homology between mouse and human. Functional analysis of promoter activity of the 5'-flanking region of cyclin D2 suggested that the region - 1,100 to - 805 including C/EBP, PEA3, AP2, NF-Y, c-Myc, and Sp1 may have a major positive regulatory activity for expression of cyclin D2.

Animals↗

PubMed1994

Cloning, expression and characterization of thymidylate synthase from Cryptococcus neoformans.

The thymidylate synthase (TS)-encoding gene from Cryptococcus neoformans (Cn) has been isolated from cDNA and genomic libraries. The 1127-bp gene contains three introns and a 951-bp open reading frame encoding a 35,844-Da protein. The cDNA clones lack 324 bp of the 5' coding region of the gene. The complete coding sequence was assembled as an expression cassette in pUC19 using parts of the coding sequence from the cDNA and genomic DNA and completing the sequence using synthetic DNA. Production of active TS from Cn (CnTS) was first demonstrated by complementation of a thymine(Thy)-requiring Escherichia coli strain. The expression cassette was subsequently subcloned into the T7 polymerase vector pET15-b. In this construct, CnTS is produced as approximately 10% of the total soluble protein in E. coli. Homogeneous enzyme was obtained at a 36% yield after consecutive chromatography on DEAE-cellulose, Q-Sepharose, phenyl-Sepharose and Affi-Gel Blue. Steady-state kinetic analysis showed that the Km values for dUMP and CH2H4.folate were 2.7 +/- 0.5 microM and 38.2 +/- 2.5 microM, respectively, and the kcat was 5.1 s-1. The enzyme was stable upon storage at -80 degrees C in Tris.HCl pH 7.4 and thiol.

Amino Acid Sequence↗

PubMed2000

Encephalitozoon cuniculi (Microspora) genome: physical map and evidence for telomere-associated rDNA units on all chromosomes.

A restriction map of the 2.8-Mb genome of the unicellular eukaryote Encephalitozoon cuniculi (phylum Microspora), a mammal-infecting intracellular parasite, has been constructed using two restriction enzymes with 6 bp recognition sites (Bss HII and Mlu I). The fragments resulting from either single digestions of the whole molecular karyotype or double digestions of 11 individual chromosomes have been separated by two-dimensional pulsed field gel electrophoresis (2D-PFGE) procedures. The average distance between successive restriction sites is approximately 19 kb. The terminal regions of the chromosomes show a common pattern covering approximately 15 kb and including one 16S-23S rDNA unit. Results of hybridisation and molecular combing experiments indicate a palindromic-like orientation of the two subtelomeric rDNA copies on each chromosome. We have also located 67 DNA markers (clones from a partial E. cuniculi genomic library) by hybridisation to restriction fragments. Partial or complete sequencing has revealed homologies with known protein-coding genes for 32 of these clones. Evidence for two homologous chromosomes III, with a size difference (3 kb) related to a subtelomeric deletion/insertion event, argues for diploidy of E.cuniculi. The physical map should be useful for both the whole genome sequencing project and studies on genome plasticity of this widespread parasite.

Animals↗

PubMed2000

Presence of a pertussis toxin-sensitive G protein alpha subunit in Sporothrix schenckii.

As an initial step in the study of the role of G proteins in signal transduction in Sporothrix schenckii, we identified a Galphai subunit using different experimental approaches. Western blots of fungal membrane preparations using anti-Galphacommon and anti-Galphai1-Galphai2 antibodies identified a band of approximately 41 kDa. Pertussis toxin-catalyzed adenosine diphosphate (ADP)-ribosylation of these membrane fractions confirmed the presence of a protein substrate of 41 kDa. A 357 bp polymerase chain reaction (PCR) product obtained using fungal DNA as template and primers targeted to conserved Galphai sequences, was used as a probe to isolate a clone from an S. schenckii genomic library. A partial sequence for a Galphai subunit was obtained from this clone. The sequence was completed using the rapid amplification of cDNA ends (RACE) technique with mycelium and yeast cDNA. The cDNA sequence revealed a 1059 bp open reading frame encoding a 353 amino acid Galphai subunit of 41 kDa, more than 90% identical to the CPG-1 of Cryphonectria parasitica, and GNA-1 of Neurospora crassa. The genomic sequence was obtained by PCR using fungal DNA, and revealed a 1250 bp sequence and the presence of three introns. These results provide evidence for the first time of the presence and expression of a Galphai homolog in a pathogenic dimorphic fungus.

Amino Acid Sequence↗

PubMed1997

Isolation of synthetic lethal mutants of ras1 in Schizosaccharomyces pombe.

Ras proteins are membrane-associated guanine nucleotide-binding proteins that serve as molecular switches for signal transduction pathways in a diverse array of organisms. Various cellular factors are known to interact with Ras proteins. In order to find the novel cellular factors that are associated with Ras function, we have constructed synthetic lethal mutants of the ras1+ gene in Schizosaccharomyces pombe and used them to identify the genes that are functionally dependent on the Ras1. We first constructed S. pombe strains in which chromosomal ras1+ gene is placed under the nmt1 promoter that is regulated by thiamine. This strain shows ras1+ phenotype in the absence of thiamine, whereas it shows ras1- phenotype in the presence of thiamine. Second, we mutated the constructed strains with ultraviolet light (UV) and selected two synthetic lethal mutants that could not grow when Ras1 function was repressed (ras1-). One of the mutants, KSC3, showed a swollen cell shape, aberrant deposition of septum materials, and aberrant nuclei. The other mutant, KSC4, showed sensitivity to hyper-osmolarity when Ras1 function is absent. These mutants, however, grow normally when Ras1 is expressed (ras1+). These two novel synthetic lethal mutants of ras1 provide the means to isolate the corresponding genes that function in association with Ras1 in S. pombe. Screening of a genomic library of S. pombe complementing the mutant phenotype allowed us to identify several novel genes associated with Ras1 of S. pombe.

Cell Size↗

PubMed1989

DNA length variants contiguous to the 3' end of a vitellogenin gene in Drosophila grimshawi laboratory stocks from different Hawaiian Islands.

Two V3 vitellogenin clones isolated from genomic libraries of Drosophila grimshawi (G1, Auwahi, Maui) were found to differ in length. Structural comparison of the two clones established that the length difference could be attributed to two insertions/deletions of about 200 bp each, both within the 3' flanking sequences of the gene. The two length variants appeared to be polymorphic in the G1 laboratory strain, as demonstrated by analysis of genomic DNA isolated from single flies. The deleted variant sequence was traced by further analysis to two other D. grimshawi strains (PK9 and S10G1) which originated from the island of Molokai. The existence of this morph in the Maui strain appears to have resulted from a laboratory stock contamination at the Drosophila Stock Center. In the course of a few generations of culture of this G1 strain at New York University, the deleted morph increased its frequency surprisingly rapidly, almost replacing the original morph, while at the Bowling Green Stock Center, the original morph still predominates. These frequency changes are most likely consequences of genetic drift due to bottlenecks in the maintenance and propagation of this stock.

Animals↗

PubMed1994

Restoration of pathogenicity of a penetration-deficient mutant of Collectotrichum lagenarium by DNA complementation.

Infection by Colletotrichum lagenarium requires formation of an appressorium and of a penetration peg. A mutant, 83,348, defective in morphogenesis of the penetration peg was unable to penetrate into cellulose membranes or infect cucumber leaves. DNA transformation using a wild-type genomic library constructed in pKVB resulted in two transformants, Ppr1 and Ppr2, with restored penetration peg formation, from 2,000 benomyl-resistant transformants. However, penetration into cellulose membranes by these transformants ranged from 30 to 40% compared to greater than 90% by wild-type. Southern-blot hybridization showed that a single copy of a cosmid clone had integrated into the genome of the transformants. A 12.0-kbp fragment of the cosmid vector with the flanking region of wild-type genomic DNA was recovered by plasmid rescue from Ppr1. Using the flanking DNA sequences as a probe for colony blot hybridization, a genomic clone was identified and designated pRP46. Transformants obtained following transformation with pRP46 were able to penetrate cellulose membranes. The penetration frequency of pRP46 transformants ranged from 25 to 65%. Transformants were also pathogenic on cucumber.

Cloning, Molecular↗

PubMed1996

Prolactin-like protein-C variant: complementary deoxyribonucleic acid, unique six exon gene structure, and trophoblast cell-specific expression.

The rat placental PRL family consists of proteins structurally related to pituitary PRL. As a consequence of attempting to characterize the gene for one of the members of the family, PRL-like protein-C (PLP-C), we identified a related gene that we have termed PLP-C variant (PLP-Cv). In this study, we present information on the PLP-Cv gene and its pattern of expression. Screening of a rat genomic library with a PLP-C cDNA resulted in the isolation of four phage clones. Nucleotide sequence analysis of the clones revealed a gene, PLP-Cv, closely related but distinct from PLP-C. The PLP-Cv gene possessed a six exon/five intron organization, unique among members of the PRL family, and was localized to chromosome 17 of the rat genome, similar to other PRL family members. A PCR strategy involving primers based on the PLP-Cv gene was used to isolate a placental PLP-Cv cDNA. PLP-Cv showed 90 and 78% sequence identity with PLP-C at nucleotide and amino acid levels, respectively. Expression of PLP-Cv was restricted to the trophoblast lineage and was coordinately activated with PLP-C beginning at day 11 of gestation and continuing until term. Primer extension analysis revealed multiple putative transcription start sites. A 2.1-kilobase pair PLP-Cv promoter-luciferase reporter construct was specifically activated in differentiating rat trophoblast cells but not in other cell types. In conclusion, we have identified a new member of the PRL family possessing considerable homology to PLP-C, a unique gene structure, and displaying a trophoblast-specific pattern of transcriptional activation.

Amino Acid Sequence↗

PubMed2000

From a short amino acidic sequence to the complete gene.

A useful strategy directed to the isolation of a required gene with a high GC content is reported. Using a degenerate oligonucleotide probe, deduced from the amino terminus of a protein, it is possible to obtain a fragment of DNA containing its encoding gene by PCR amplification. Furthermore, the cloning of a desired gene can be accomplished in two steps by using an oligonucleotide deduced (i) from an internal sequence, (ii) from a consensus sequence, or (iii) from a DNA sequence adjacent to a disrupting element (transposon, insertion sequence, cassette). This method, which could be applied to a bacteriophage, plasmid, or cosmid genomic library, has been successfully used for cloning several genes from different biological systems.

Amino Acid Sequence↗

PubMed1990

Human ornithine decarboxylase-encoding loci: nucleotide sequence of the expressed gene and characterization of a pseudogene.

Previous studies have shown that human ornithine decarboxylase (ODC)-encoding sequences map to two chromosome regions: 2pter-p23 and 7cen-qter. In the present work we have cloned the expressed human ODC gene from a genomic library of myeloma cells that overproduce ODC protein due to selective gene amplification and determined its entire nucleotide sequence. The gene comprises 12 exons and 11 introns and spans about 8 kb of chromosome 2 DNA. The organization of the human gene is very similar to that of the mouse and rat, with the major difference being the presence of longer intronic sequences in the human gene. Some of these differences can be accounted for by the insertion of four Alu sequences in the human gene. Several potential regulatory elements are present in the promoter region and in 5'-proximal introns, including a TATA box; GC boses; AP-1-, AP-2- and NF-1-binding sites; and a cAMP-responsive element. The 5'-untranslated sequence of ODC mRNA is extremely GC-rich, and computer predictions suggest a very stable secondary structure for this region, with an overall free energy of formation of -225.4 kcal/mol. In addition to the active ODC gene on chromosome 2, ODC gene-related sequences were isolated from human chromosome 7-specific libraries and shown to represent a processed ODC pseudogene.

Animals↗

PubMed1995

Molecular cloning of the leuB gene from Bacteroides fragilis by functional complementation in Escherichia coli.

Clones containing the Bacteroides fragilis leuB-complementing gene were isolated by screening of a B. fragilis genomic library constructed in Escherichia coli. One recombinant clone, designated pOT865, with the smallest DNA insert (4.5 kb) could complement three independent leuB mutations in E. coli and the leuB-complementing determinant in pOT865 was localized to a region of 1.5-kb DNA. The results of Southern blot analysis suggested that a single copy of the cloned gene was present in the B. fragilis genome. The cloned fragment appeared to contain a sequence that could function as promoter in E. coli and direct the synthesis of a 42-kDa protein. These results suggest that the cloned segment contains the structural gene for beta-isopropylmalate dehydrogenase (leuB).

3-Isopropylmalate Dehydrogenase↗

PubMed1991

Regional mapping of 22 microclones around the adenomatous polyposis coli (APC) locus on chromosome 5q.

A previously described genomic library constructed from microdissected DNA has been used to generate a large number of probes around the adenomatous polyposis coli (APC) gene at 5q22. A total of 202 clones were hybridised directly onto a somatic cell hybrid panel containing two APC-related interstitial deletions. Of 75 microclones that gave clear hybridisation signals, 22 independent clones mapped into the region common to both deletions. In addition, 4/22 of the markers are conserved in rodent DNA. These clones should provide a valuable resource for screening cDNA libraries and cloning the DNA around the APC gene in yeast artificial chromosomes.

Adenomatous Polyposis Coli↗

PubMed1999

Mytilus edulis histone gene clusters containing only H1 genes.

We isolated five different phage clones containing histone gene clusters with up to five H1 genes per phage clone from a Mytilus edulis genomic library. Among these H1 genes, nine gene types coding for five different H1 proteins have been identified. All H1 histone genes were located on repetitive restriction fragments with only slightly different sizes. The H1 coding regions show highly related sequences, suggesting that the multitude of H1 genes has evolved by gene duplication events. Core histone genes could not be found on these five Mytilus edulis genome fragments.

Amino Acid Sequence↗

PubMed1991

A portion of the human surfactant protein A (SP-A) gene locus consists of a pseudogene.

SP-A is the most abundant, surfactant-associated protein isolated from lung lavage. Genomic blot analysis of total human cellular DNA with SP-A cDNA demonstrated the presence of multiple hybridizing fragments that are not accounted for by available SP-A gene sequences. In this report, we have cloned and characterized human genomic DNA fragments that account for some of the other hybridizing fragments. These clones contain nucleotide sequences that are highly homologous to the fourth intron and fifth exon of the human SP-A gene. Sequences upstream from these SP-A-like sequences are not detectable by Northern blot hybridization of SP-A-expressing cells and the SP-A-like sequences contain premature stop codons, consistent with the interpretation that these clones represent an SP-A pseudogene. Restriction fragments consistent with this pseudogene and the functional SP-A gene are present in a human chromosome 10 genomic library made from a single chromosome, showing that the functional SP-A gene and the pseudogene are syntenic.

Bacteriophage lambda↗

PubMed1990

Isolation and characterization of a developmentally regulated homeobox sequence in the Mexican axolotl Ambystoma mexicanum.

A homeobox-containing genomic DNA fragment was isolated from the Mexican axolotl. This clone was obtained from a partial genomic library enriched for sequences that cross-hybridized with the Drosophila Antp homeobox under low stringency hybridization conditions. DNA sequence analysis revealed that this sequence (Ahox1) was 66% homologous to the Antp homeobox sequence and was most closely related to the mouse Hox-1.6 (84% identity) and Drosophila lab (79% identity) homeobox sequences. Several cross-hybridizing fragments to Ahox1 were detected in both mouse and axolotl genomic DNA. This sequence was also shown to be conserved in other Ambystoma species. Northern blot analysis revealed that genes containing this sequence are developmentally regulated. Transcripts hybridizing to the Ahox1 homeobox probe were detected during the neurula and tail bud stages of development.

Ambystoma↗

PubMed1995

Identification of mycobacterium tuberculosis DNA sequences encoding exported proteins by using phoA gene fusions.

The activity of bacterial alkaline phosphatase (PhoA) is dependent on it being exported across the plasma membrane. A plasmid vector (pJEM11) allowing fusions between phoA and genes encoding exported proteins was constructed to study protein export in mycobacteria. Introduction of the Mycobacterium fortuitum beta-lactamase gene (blaF*) into this vector led to the production in M. smegmatis of protein fusions with PhoA activity. A genomic library from M. tuberculosis was constructed in pJEM11 and screened in M. smegmatis for clones with PhoA activity. Sequences of the M. tuberculosis inserts directing the production of protein fusions in these PhoA-positive clones were determined. They include part of the already-known exported 19-kDa lipoprotein, a sequence with similarities to the exported 28-kDa antigen from M. leprae, a sequence encoding a protein sharing conserved amino acid motifs with stearoyl-acyl-carrier-protein desaturases, and unknown sequences. This approach thus appears to identify sequences directing protein export, and we expect that more extensive screening of such libraries will lead to a better understanding of protein export in M. tuberculosis.

Alkaline Phosphatase↗

PubMed1993

Five skeletal myosin heavy chain genes are organized as a multigene complex in the human genome.

Myosin heavy chain (MyHC) isoforms are encoded by a multigene family in vertebrates. We used genomic DNA mapping by pulse field gel electrophoresis to demonstrate that, in humans, the embryonic, fetal, fast IIB and IIX MyHC genes and a gene coding for a non-identified striated muscle MyHC fast-type isoform (NI), are contained within a 320 kb SalI genomic fragment. The locus is flanked by two CpG islands, separated by 580 kb. In order to further characterize the MyHC genes, a human genomic library constructed in yeast artificial chromosomes (YAC) was screened and five independent clones were isolated. Characterization of these YACs revealed that one of them contains at least five MyHC genes, based on partial sequencing of their conserved third coding exons. Three of these genes correspond to those encoding the embryonic, fetal and fast IIB MyHC isoforms. Moreover, in this YAC clone the embryonic and fetal genes, on the one hand, and the adult fast (IIB, IIX and NI) genes, on the other hand, are contained within two different ClaI fragments. This result suggests that the genes encoding the two developmental forms are adjacent in the human genome and that temporal regulation of the MyHC genes might be related to their organization within the locus. These data represent the first direct evidence for the existence in the human genome of a MyHC multigene locus that contains at least five genes.

Amino Acid Sequence↗

PubMed1994

An oligogene family encodes actins in the housefly, Musca domestica.

Southern hybridization analyses of genomic DNA and dot-blot reconstruction experiments indicated that 5-6 members comprise the actin gene family in the housefly, Musca domestica. Hybridization data failed to reveal conservation between housefly and fruitfly sequences flanking actin genes at their 3' ends, in marked contrast to the high degree of conservation observed in these regions of orthologous actin genes in vertebrates. Hybridization analysis of genomic DNA isolated from individual houseflies revealed polymorphism within actin-associated sequences. A representative genomic library was constructed in lambda EMBL3 and screened using a heterologous actin coding sequence probe. Of nearly 20 recombinants isolated, 6 represented overlapping clones and the remainder proved to be inviable.

Actins↗