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PubMed1993

Characterization, cloning and expression of the 67-kDA annexin from chicken growth plate cartilage matrix vesicles.

Analysis of a 67 kDa lipid-dependent Ca(2+)-binding protein from avian matrix vesicles revealed amino acid sequences homologous to mammalian annexin VI. PCR methods were used to identify a clone from an avian cDNA library that contained a full length copy of the 67-kDa annexin cDNA. This was restriction mapped, subcloned and sequenced. The cDNA sequence of the open reading frame showed 70 percent identity to that of murine annexin VI; the predicted amino acid sequence, 78 percent identity. There was no homology in the 5'- and 3'-untranslated regions. A plasmid was constructed that overexpresses the intact chicken 67-kDa matrix vesicle annexin in E. coli DH5 alpha in high yield; the physicochemical properties and the amino terminal sequence of the expressed protein exactly matched those of the native protein.

Amino Acid Sequence↗
PubMed2000

In vivo conformation of mitochondrial DNA revealed by pulsed-field gel electrophoresis in the true slime mold, Physarum polycephalum.

Pulsed-field gel electrophoresis (PFGE) was used to examine the in vivo and in vitro conformations of Physarum polycephalum mitochondrial DNA (mtDNA). We used plugs containing isolated mitochondria, isolated mitochondrial nucleoids (mt-nuclei), and isolated mtDNA, in addition to whole cells. The mtDNA contained in the myxamoebae, plasmodia, isolated mitochondria, and isolated mt-nuclei was circular, but most of the isolated mtDNA had been site-specifically fragmented and linearized during DNA preparation and storage under low ionic strength conditions. Restriction mapping of Physarum mtDNA by the direct digestion of the isolated mt-nuclei from two different strains, DP89 x AI16 and KM88 x AI16, resulted in the circular form. A linear mitochondrial plasmid, mF, is known to promote mitochondrial fusion and integration of itself into the mtDNA in Physarum. Linearization of mtDNA by the integration of the mF plasmid was demonstrated when we used PFGE to analyze isolated mitochondria from the plasmodial strain DP89 x NG7 carrying the mF plasmid (mF+). The PFGE system can be used not only to determine whether the form of mtDNA is linear or circular but also to analyze the dynamic conformational changes of mtDNA.

Animals↗
PubMed1988

Extra-ribosomal spacer sequences in Triturus.

We show that, in Triturus vulgaris meridionalis, sequences homologous to the rDNA "non-transcribed" spacer (NTS) are clustered at chromosomal loci where they are not associated with 18 S or 28 S rDNA genes: these sequences are referred to as the extra-ribosomal spacer sequences. Genomic clones containing such extra-ribosomal spacer sequences have been isolated. As shown by restriction mapping, these clones appear to consist mostly of repetitive BamHI fragments that are, in turn, internally repetitious and highly homologous to each other. The structure of the clones was confirmed by nucleotide sequence analysis, which also demonstrates the high degree of conservation between the BamHI elements and the homologous NTS sequences. An intriguing 12 base-pair homology between the extra-ribosomal spacer sequences and a Xenopus NTS enhancer sequence is reported. The possibility that a repetitive octanucleotide motif found within the BamHI elements could act as a recombination hotspot by virtue of its similarity with the Escherichia coli chi sequence is discussed.

Animals↗
PubMed1993

Use of a PCR-based method to characterize protein kinase C isoform expression in cardiac cells.

Molecular cloning has identified at least nine unique isozymes of protein kinase C (PKC) designated alpha, beta I, beta II, gamma, delta, epsilon, zeta, and eta/L, with the recent addition of the theta-isoform. Previous attempts to characterize PKC isoform expression in heart have been limited by low levels of protein and perhaps by the presence of novel isoforms. Thus to critically examine the diversity of PKC expression in cardiac cells, we developed a reverse transcriptase-polymerase chain reaction (RT-PCR) approach that would amplify regions of the target cDNA of all the PKC isozymes in a single reaction. Degenerate oligonucleotide primers were designed to recognize sequences in the conserved regions of the PKC sequence motif: the cysteine-rich and the ATP-binding regions. Amplification of target PKC cDNA sequences resulted in PCR products with unique sizes and restriction digestion properties. The system was validated by identifying PCR products that correspond to all of the PKC isoform transcripts, except PKC-zeta, in a single reaction with cDNA derived from hippocampus. Cardiac cDNA was RT-PCR amplified, and the products were analyzed by a combination of restriction mapping and DNA sequencing that revealed the presence of only the alpha, delta, epsilon, and eta isoforms in adult rat cardiac myocytes and cultured neonatal ventricular myocytes. A unique nondegenerate primer pair was synthesized to recognize PKC-zeta cDNA. Results with these primers show that PKC-zeta is present in both cardiac myocyte preparations as well. The RT-PCR method developed here is an efficient approach that is broadly useful to examine PKC expression in many tissues, including the identification of potentially novel isoforms.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1988

Transcriptional regulation and gene arrangement of Escherichia coli, Citrobacter freundii and Salmonella typhimurium biotin operons.

The bio operons of Citrobacter freundii and Escherichia coli K-12 (strain C600) were isolated by screening lambda banks for complementation of E. coli bio mutants. These were compared with the previously isolated bio operon of Salmonella typhimurium and previous data on E. coli K-12. The restriction maps of the operon are very different in the three species, but no difference in gene order was found. Operator-promoter DNA, identified by repressible titration and by biotin-repressible transcription in E. coli, was sequenced and compared to the published E. coli K-12 sequence. In the segment previously identified as operator/bioB promoter, C. freundii and S. typhimurium DNA are identical and differ from E. coli only by 2 bp. The DNA to the right of this segment (indicated by previous data to be the bioA promoter of E. coli) has diverged in all three species, and only E. coli has a sequence resembling a consensus promoter.

Bacteriophage lambda↗
PubMed1990

Major histocompatibility complex-linked diabetogenic gene of the nonobese diabetic mouse. Analysis of genomic DNA amplified by the polymerase chain reaction.

Inheritance of insulin-dependent diabetes mellitus (IDDM) is polygenic, and at least one of the genes conferring susceptibility to diabetes is tightly linked to the MHC. Recent studies have suggested that DQB1 of humans and I-A beta of mice are closely associated with susceptibility and resistance to IDDM. For further characterization and localization of the MHC-linked diabetogenic gene, we studied the genomic sequence of the A beta gene of the nonobese diabetic (NOD) mouse, an animal model of IDDM, in comparison with those of its sister strains, nonobese nondiabetic and cataract Shionogi (CTS) mice, and the original strain, outbred Imperial Cancer Research (ICR) mice. Genomic DNAs from these strains were amplified in vitro by the polymerase chain reaction with thermostable Taq polymerase. The amplified sequences were analyzed by restriction endonuclease digestion, hybridization with allele-specific oligonucleotide probes, and direct sequencing. The unique I-A beta sequence of NOD mice was observed in the sister strain, CTS mice, and in one mouse of the original strain, outbred ICR mice. These data together with the results of MAb typing of MHC molecules and restriction mapping of the I-A region suggest that the unique class II MHC of NOD mice is not the result of a recent mutation, but is derived from the original strain. Since class I MHC of CTS mice is different from the MHC of NOD mice at both the K and D loci, CTS mice are a naturally occurring recombinant strain with NOD type class II MHC and non-NOD type class I MHC. Thus, breeding studies in crosses of NOD with CTS mice should provide biological information on whether the unique class II MHC of NOD mice is diabetogenic.

Alleles↗
PubMedDate not supplied

Construction of an expression cassette with hTNF-alpha gene for transient expression of the gene in mammalian cells.

BACKGROUND: Tumor vaccines, which are created by the insertion of cDNA encoding different cytokines into the tumor cells, are capable of inducing a very complex immune reaction including activation of CD8 T cells, granulocytes, macrophages, the triggering of cytokine cascades and antibody production. Aiming to create genetically modified tumor cells which could produce and secrete Human Tumor Necrosis Factor-alpha (hTNF-alpha), we constructed the expression cassette containing hTNF-alpha gene in pcDNA3 plasmid vector. MATERIALS AND METHODS: The successful ligation of cDNA encoding for hTNF-alpha into pcDNA3 plasmid vector was confirmed by PCR, restriction mapping and sequence determination. The constructed expression cassette in pcDNA3 vector was than transferred in vitro into malignant melanoma B16 tumor cells by the method of Receptor Mediated Gene Transfer (RMGT). RESULTS: Measurable amounts of hTNF-alpha protein detected in the medium of transfected cells proved that tumor cells modified in this manner became producers of hTNF-alpha protein. CONCLUSION: The expression of the transferred gene was transient and the produced protein was biologically active. Furthermore, the production of hTNF-alpha protein was also observed in sub-lethally irradiated tumor cells, showing that the expression cassette was preserved during the irradiation and that the cells were potentially applicable as a tumor vaccine.

Animals↗
PubMed2001

Polymorphism of the virulence regulon and allelic variations of the sic gene among the emm1 isolates of group A Streptococcus from western Norway.

With the objective of finding genetic markers of invasiveness, 43 isolates of group A streptococcus, isolated in western Norway and from both severe invasive disease and superficial infections, were studied initially by restriction fragment length polymorphism of the virulence regulon (virR -RFLP). Polymorphism that seemed to be related to the severity of infection was observed within the emm1 sequence type, which included 11 invasive and seven non-invasive isolates. These emm1 isolates were further investigated by restriction mapping of the virR and sequence analysis of a polymorphic region, which revealed the presence of a hypervariable sic gene. Of the nine distinct sic alleles, seven were found in single isolates, of which only two were from patients with invasive disease. The other two alleles were shared among nine invasive and two non-invasive isolates. The presence of only two sic allotypes in nine of the 11 invasive isolates, as compared to a different allele in each of the five non-invasive, contemporary isolates supports the hypothesis that selection of the sic variants occurs at mucosal surfaces and implicates mainly two clones among the invasive emm1 isolates.

Alleles↗
PubMed1998

The major APN transcript of the alveolar type II epithelial cell originates from a unique upstream promoter region.

Aminopeptidase N (APN, EC 3.4.11.2) is an ectopeptidase expressed in lung at the apical surface of alveolar type II epithelial cells. Its expression is upregulated during fetal lung development. To begin to understand the regulation of APN expression during lung development, we used the rapid modification of cDNA ends (RACE) to clone the 5' end of the major APN transcript in rat lung and alveolar type II cells. The cloned sequence revealed a unique 135 bp untranslated exon which genomic cloning and restriction mapping indicated was located more than 14 kb upstream from the coding sequence. A 172 bp genomic fragment flanking the untranslated exon produced a high level of expression of a reporter gene in transient transfection assays using a human lung adenocarcinoma cell line. The DNA fragment includes elements known to be important for expression of lung specific proteins, including the surfactant-associated proteins A, B, and C and the Clara cell specific protein. Comparison of the APN genomic sequences and gene structure from human and rat suggests that the exon present in the rat lung transcript may result from the use of a previously uncharacterized APN promoter.

Animals↗
PubMed1993

High-frequency genetic reversion mediated by a DNA duplication: the mouse pink-eyed unstable mutation.

The mouse pink-eyed unstable (p(un)) mutation, affecting coat color, exhibits one of the highest reported reversion frequencies of any mammalian mutation and is associated with a duplication of genomic DNA at the p locus. In this study, genomic clones containing the boundaries of the p(un) duplication were isolated and characterized. The structure of these sequences and their wild-type and revertant counterparts were analyzed by restriction mapping, PCR product analysis, DNA sequence analysis, and pulsed-field gel electrophoresis. DNA from p(un) was distinguished from wild-type and revertant DNA by a head-to-tail tandem duplication of approximately 70 kilobases. No differences were detected between revertant and wild-type DNAs. Thus, the reversion in phenotype of p(un) mice is coupled with the loss of one copy of an approximately 70-kilobase duplicated segment. Testable models are presented to account for p(un) reversion.

Alleles↗
PubMed1990

Mode of action and substrate specificity of a purified exo-1,4-beta-D-glucosidase cloned from the cellulolytic bacterium Ruminococcus albus AR67.

A gene encoding exo-1,4-beta-D-glucosidase, from Ruminococcus albus AR67, was cloned in Escherichia coli, restriction mapped, and shown to be expressed from sequences within the insert that function as a promoter in E. coli. The cloned enzyme was located predominantly in the cytoplasm (40%) and attached to insoluble cell components (48%). After purification to homogeneity, the enzyme (Mr = 64,000, monomeric) was specific for substrates with beta-D-glucopyranosyl configuration and was inactive against alpha-glucosides, lactosides and xylosides. Km values of the enzyme decreased with increasing chain length (G2-G5). Glucose was the major product of hydrolysis from cellodextrins. Preference for longer chain cellodextrins is consistent with exo-1,4-beta-D-glucan glucohydrolase mode of action [E.C. 3.2.1.74].

Chromatography, Gel↗
PubMed1989

Molecular analysis of one of multiple protease-encoding genes from the prototype virulent strain of Bacteroides nodosus.

The aim of these studies was to examine the organization of the Bacteroides nodosus protease-encoding gene(s). The extracellular serine proteases (38 kDa) from the prototype virulent strain of B. nodosus were purified and used to raise a specific antiserum in rabbits. This antiserum was used in a colony immunoassay to screen a genomic DNA library constructed in Escherichia coli using BamHI-digested B. nodosus DNA and the plasmid pBR322. An E. coli clone expressing a 50-kDa immunoreactive polypeptide was identified. No protease activity was detected in the culture media, or in crude soluble and membrane fractions prepared from this clone. Restriction mapping and deletion analysis of the recombinant plasmid, pEKM2, was used to locate the coding region to a 1.4-kb EcoRI-BamHI fragment which was subsequently sequenced. A large open reading frame was found to extend through the BamHI site from a putative start codon just downstream from the EcoRI site, which indicated that the complete gene was not isolated. Southern blotting demonstrated that there were at least three B. nodosus BamHI fragments which were homologous to the 0.4-kb PstI-BamHI fragment of pEKM2. Based on these results the existence of multiple protease genes in B. nodosus was postulated.

Amino Acid Sequence↗
PubMed1992

Rat copper/zinc superoxide dismutase gene: isolation, characterization, and species comparison.

A 13 kb rat Cu/ZnSOD genomic clone has been purified from a rat liver genomic library and completely characterized by restriction mapping, detailed sequencing and Southern blot analysis. This gene spans approximately 6 kb and contains five exons and four introns. Comparison of rat, mouse, and human Cu/ZnSOD genes reveals a high conservation in genomic organization and exon-intron junctions, including an unusual 5'GC donor sequence at the first intron. The gene contains a TATA box as well as an inverted CCAAT box, a feature common to both the mouse and human genes. Furthermore, several repeats were identified in the 5' promoter region of this gene, and these regulatory elements are also strikingly conserved in these three species.

Amino Acid Sequence↗
PubMed1992

Structure of the human TNF receptor 1 (p60) gene (TNFR1) and localization to chromosome 12p13 [corrected].

Clones encoding the entire coding and 3' untranslated region of the human type I tumor necrosis factor receptor (p60) gene (TNFR1) were isolated by hybridization using probes derived from TNFR-1 cDNA. The gene was characterized by restriction mapping. DNA blot analysis and sequence analysis. The coding region and the 3' untranslated region are distributed over 10 exons. Each of the four repeats, comprising the extracellular ligand binding domain and characterizing a receptor superfamily, is interrupted by an intron. However, the intron-exon structure is not conserved in the nerve growth factor receptor gene, another member of this superfamily. By PCR analysis of human-mouse somatic cell hybrids and in situ hybridization using biotinylated genomic TNFR1 DNA, we localized the gene to human chromosomal band 12p13. This corresponds to the homologous murine gene localized at the distal region of mouse chromosome 6.

Amino Acid Sequence↗
PubMed1998

The normal copy of the G0S19-3-associated, CpG island-containing, upstream sequence is downstream of G0S19-2/MIP1alpha in association with a TRE17 oncogene.

The G0S19-1/MIP1alpha and G0S19-2/MIP1alpha genes locate to human chromosomes 17q and encode similar copies of the beta-chemokine G0S19/MIP1alpha. The G0S19-3 gene, present in 1 in 4 humans, is a 5' truncated version of G0S19-2; a CpG island-containing upstream sequence (CpG-US), rich in potential transcriptional activation motifs, replaces much of the first intron and the first exon. Sequences hybridizing with the CpG-US sequence, normally exist in all human genomes. Thus, it appears that there has been recombination between a duplicated G0S19 gene and a duplicated CpG-US-like sequence. We have isolated sequences hybridizing with the CpG-US sequence from a human genomic library in bacteriophage lambda. Restriction mapping and sequencing shows a CpG-US-like sequence approximately 8 kb downstream of G0S19-2 (hence, named CpG-DS sequence). The sequence is contiguous with a TRE17 oncogene-associated sequence (GenBank locus HSTRE175). Members of the TRE17 family are known to locate to chromosome 17q (Onno et al., 1993b), and have sequence characteristics suggestive of positive Darwinian selection. Linkage with a TRE17 oncogene may have arisen by recombination and imply no functional relationship. However, it is possible that the CpG-DS may normally regulate TRE17 expression. PCR and sequencing studies indicate the close proximity of other chemokine-related sequences in the 17q11.2 region.

Amino Acid Sequence↗
PubMed1993

Linear mitochondrial DNAs of yeasts: frequency of occurrence and general features.

In most yeast species, the mitochondrial DNA (mtDNA) has been reported to be a circular molecule. However, two cases of linear mtDNA with specific termini have previously been described. We examined the frequency of occurrence of linear forms of mtDNA among yeasts by pulsed-field gel electrophoresis. Among the 58 species from the genera Pichia and Williopsis that we examined, linear mtDNA was found with unexpectedly high frequency. Thirteen species contained a linear mtDNA, as confirmed by restriction mapping, and labeling, and electron microscopy. The mtDNAs from Pichia pijperi, Williopsis mrakii, and P. jadinii were studied in detail. In each case, the left and right terminal fragments shared homologous sequences. Between the terminal repeats, the order of mitochondrial genes was the same in all of the linear mtDNAs examined, despite a large variation of the genome size. This constancy of gene order is in contrast with the great variation of gene arrangement in circular mitochondrial genomes of yeasts. The coding sequences determined on several genes were highly homologous to those of the circular mtDNAs, suggesting that these two forms of mtDNA are not of distant origins.

Base Sequence↗
PubMed1999

The mitochondrial genome of Mucor piriformis.

DNA was purified from the isolated mitochondria of a Mucor piriformis wild-type strain (NRRL 26211). A circular restriction map of the mitochondrial DNA was established on the basis of single and double digests with several restriction endonucleases. The average mitochondrial DNA size calculated from these data was 33.53 kbp; this is in good agreement with the contour length size (33.62 kbp) of the open circular molecules detected by electron microscopy. Heterologous hybridizations with cloned Aspergillus nidulans mitochondrial genes were used to locate some coding regions on the map.

DNA, Fungal↗
PubMed1990

Isolation and characterization of comL, a transcription unit involved in competence development of Bacillus subtilis.

Using the transformation-deficient mutant M465, which was previously isolated by means of insertional mutagenesis with plasmid pHV60, a transcription unit comL required for genetic competence of Bacillus subtilis was identified. A chromosomal DNA fragment flanking the inserted pHV60 was isolated and used to screen two different libraries of B. subtilis DNA in phage lambda EMBL4 and lambda EMBL12, respectively. With the aid of six recombinant phages that hybridize with this chromosomal fragment a restriction map of about 23 kb of B. subtilis chromosomal DNA was constructed. Using small adjoining pieces of this chromosomal DNA in Campbell integrations, the size of the transcription unit involved in competence development could be delimited to about 15 kb. By insertion of a promoterless lacZ gene into comL, the transcriptional regulation of comL was analysed and epistatic interactions among various other com genes were determined. The results of these experiments indicated that comL is optimally expressed in glucose-based minimal medium when the culture enters the stationary phase of growth and that the expression of late competence genes is dependent on previous transcription of comL, which in turn is dependent on the gene products of comA and comB.

Bacillus subtilis↗