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PubMed1996

Swine cytosolic malic enzyme: cDNA cloning, sequencing, and localization.

A highly significant genetic association has been found between some alleles of the swine Major Histocompatibility Complex SLA (Swine Leukocyte Antigen genetic complex) and the cytosolic malic enzymatic activity level in muscles. The aim of this study was to find out whether this genetic association was due to a close linkage of the SLA region and the gene coding for the enzyme. Since no swine cytosolic malic enzyme sequence (ME1) was available, we isolated several overlapping fragments that spanned the almost entire malic enzyme transcript both by screening of a swine cDNA library and by RT-PCR. The results indicated the existence of two transcripts of 2. 0 and 3.1 kb, which probably correspond to two alternative forms of one gene. The sequence of the transcript was highly similar to the other published mammalian cytosolic NADP+-dependent malic enzyme cDNA, especially within the four functional domains. Two major bands at 3.7 and 2.4 kb were detected on Northern blots containing the RNA from 25 tissues from fetuses and adult pigs. A high expression level was found in the adrenal gland, muscle, liver, and peripheral nerves. The analysis of malic enzyme RFLPs in five SLA informative families revealed an independent segregation of the ME1 gene from the SLA region. In situ hybridization results localized the cytosolic malic enzyme on the swine Chromosome (Chr) 1p1.2, except that the association between SLA and the malic enzyme activity level was due to a physical genetic linkage. Thus, the mechanisms underlying this association remain to be elucidated.

Amino Acid Sequence↗
PubMed2004

Simultaneous determination of Fe(II) and Fe(III) in pharmaceutical formulations with chromogenic mixed reagent by using principal component artificial neural network and multivariate calibration.

The use of chemometric approaches for the simultaneous determination of Fe(II) and Fe(III) ions has been explored by means of a two component reagent. Mixed reagents of 1,10-phenanthroline and thiocyanate were used as a selective chromogenic system for speciation of Fe(II) and Fe(III). Although the complexes of Fe(II) and Fe(III) with mixed reagent show a spectral overlap, they have been simultaneously determined with chemometric approaches, such as principal component artificial neural network (PC-ANN), principal component regression (PCR) and partial least squares (PLS). A set of synthetic mixtures of Fe(II) and Fe(III) was evaluated and the results obtained by the applications of these chemometric approaches were discussed and compared. It was found that the PC-ANN and PLS methods afforded better precision relatively than its of PCR. PC-ANN and PLS methods were also applied satisfactorily in determination of Fe(II) and Fe(III) in pharmaceutical samples.

Chemistry, Pharmaceutical↗
PubMed2005

Growth factor-sensitive molecular targets identified in primary and metastatic head and neck squamous cell carcinoma using microarray analysis.

Polypeptide growth factors play key roles in the processes of cell migration and invasion. In this study, we have used cDNA microarrays to identify target genes whose expression is differentially modulated by the growth factors TGFbeta and EGF. HN4 and HN12 cell lines, established from primary tumor and a lymph node metastasis arising in one patient with head and neck squamous cell carcinoma, were treated with 2nM EGF or 50pM TGFbeta for 24h and extracted RNA was used to prepare labeled cDNAs which were hybridized to NCI UniGem 2.0 cDNA microarrays containing 9128 features. Results revealed constitutive overexpression of 41 genes and underexpression of 109 genes in metastatic HN12 compared to HN4 under conditions of serum withdrawal. Furthermore, TGFbeta treatment resulted in relative upregulation of 53 genes and downregulation of 91 genes in HN12 compared with HN4, whereas cells treated with EGF showed relative upregulation of 67 genes and downregulation of 113 genes. Partial overlap was found between TGFbeta and EGF-modulated gene sets. Results were verified for a subset of each category using quantitative PCR, western blotting and zymography. The data indicate that TGFbeta and EGF differentially affect gene expression in primary and metastatic HNSCC cells, and likely contribute to the invasive properties of metastatic cells through regulation of both common and specific mediators for each growth factor.

Blotting, Western↗
PubMed2005

HPV16 E2 gene disruption and polymorphisms of E2 and LCR: some significant associations with cervical cancer in Indian women.

OBJECTIVES: We evaluated the status of the HPV16 E2 gene (disrupted or intact), nucleotide sequence alterations within intact E2 genes and LCR of HPV16 isolates in a group of CaCx cases (invasive squamous cell carcinomas, n = 81) and population controls (normal cervical scrapes, n = 27) from Indian women. METHODS: E2 disruption was detected by amplifying the entire E2 gene with single set of primers, while overlapping primers were used to determine if any particular region got selectively disrupted. Nucleotide variations in E2 and LCR were analyzed by PCR amplification followed by bi-directional sequencing. The associations between the viral factors and CaCx were analyzed using Fisher's Exact or Chi-squared test and interpreted as OR (95% CI) and P values. RESULTS: E2 disruption was significantly higher among the cases [3.38 (1.07-10.72); P = 0.02], which was maximum in the region between nucleotides 3650 and 3872 (DNA-binding region). The European (E) variant was found to be the prevalent subgroup (87.76% among cases and 96.30% among the controls), and the remaining samples were Asian-American variants. Among the E subgroup, variation at position 7450 (T > C) within the E2-binding site-IV was found to be significantly higher among the E2 undisrupted cases (21/37; 56.76%), compared to controls (5/18; 27.78%) [3.41 (1.01-11.55); P = 0.03]. CONCLUSIONS: Besides HPV16 E2 disruption, LCR 7450T > C variation within undisrupted E2 of E subgroup appears to be a major factor contributing to the risk of CaCx development in Indian women. Furthermore, polymorphisms in the E2 gene of HPV16 may not be significant for disease risk.

Adult↗
PubMed2000

Isolation, characterization and biological significance of hepatitis B virus mutants from serum of a patient with immunologically negative HBV infection.

BACKGROUND/AIM: A low-titered hepatitis B virus infection without immunological markers was identified in a prospective study in the serum of a kidney transplant recipient by PCR. The aim of this study was to analyze HBV genomes and their biological significance. METHODS: The genome was amplified in two overlapping fragments A and B. Sequencing of 22 clones of the A- and 12 clones of the B-fragment revealed a heterogeneous virus population. A consensus and a mutant sequence were computed, representing the complete sequence of the virus population. The two sequences were compared with 41 published genomes of the different HBV geno- and serotypes. RESULTS: Ninety-five point mutations and two deletions were identified. Two mutations were observed in all clones and 17 other mutations in three or more clones. The deletions were found in ten and seven of 22 clones. They were located in the C-gene and led to stop codons yielding truncated e- and/or core proteins. In vitro transfection of DNA constructs containing these deletions demonstrated a stop of HBV replication and of HBeAg expression. Cotransfection experiments demonstrated a dominant negative effect of the mutants containing the deletions. In addition, we describe new variants of naturally occurring HBsAg mutants that may cause HBV infection less detectable by standard HBsAg measurement assays. They were characterized by two point mutations which were observed in 9 of 12 and 13 of 22 clones of the S-gene. They significantly reduced the HBsAg expression in in vitro transfection experiments. CONCLUSION: We found a patient with low-titered HBV infection, with mutations of the 'a' epitope of the Santigen as well as with mutations leading to truncated core proteins which may cause a dominant negative effect.

Amino Acid Sequence↗
PubMed2002

Expression of slow skeletal myosin heavy chain 2 gene in Purkinje fiber cells in chick heart.

In avian, there are three slow skeletal myosin heavy chain (MHC) isoforms, slow skeletal MHC 1, 2, and 3. While slow skeletal MHC 3 has been characterized, slow skeletal MHC 1 and 2 are not yet fully studied. To determine the complete sequence of slow skeletal MHC 2, we isolated six overlapping cDNA clones, each encoding a portion of chick slow skeletal MHC 2, using the reverse transcription polymerase chain reaction (RT-PCR). The entire slow skeletal MHC 2 cDNA consisted of 5927 nucleotides including a 104 bp 3'-untranslated region and encoded 1941 amino acids. Using one of the cDNA clones, we made a probe for in situ hybridization. We also used immunohistochemistry to localize slow skeletal MHC 2 in skeletal and cardiac tissues. These studies showed that in addition to its expected expression in the adult chicken slow skeletal muscle, slow skeletal MHC 2 was expressed in the subendocardial cluster of cells and around the blood vessels within the ventricle of late embryos and adults. This isoform was not expressed in the myocardium throughout the life of the chicken. Based on morphological criteria as well as rich desmin expression, we concluded that the subendocardial cluster of cells were Purkinje cells. Although the physiological significance of the slow skeletal MHC expression remains elusive at this time, this MHC isoform may be used as a specific marker for Purkinje cells.

Animals↗
PubMed1995

cDNA selection from 10 Mb of chromosome 21 DNA: efficiency in transcriptional mapping and reflections of genome organization.

The technique of cDNA hybridization selection has been applied individually to 16 YAC clones mapping to various regions of the long arm of human chromosome 21. YACs represented approximately 10 Mb of non-overlapping DNA, and cDNA sources included fetal brain, whole fetus, and adult testes, thymus, liver and spleen. Sequencing, Northern analysis, RT-PCR and cDNA library screenings have been used to identify and partially characterize a non-redundant set of novel genes. A preliminary analysis strategy of the selected cDNAs has proven rapid and effective for isolation of the more highly represented genes and is suitable for survey transcriptional mapping efforts. By scaling up to screen > 1000 cDNA fragments per 100 kb of YAC DNA, more rare transcripts are identified and lead to comprehensive gene maps. Strong regional variations in transcriptional activity were observed, with gene densities ranging from < 1/2000 kb to > 1/15 kb. This effort has produced a large number of new genes of potential relevance to Down Syndrome.

Adult↗
PubMed1994

Identification of a unique group of human papillomavirus type 16 sequence variants among clinical isolates from Barbados.

The naturally occurring sequence variation of human papillomavirus type 16 (HPV-16) was analysed by direct sequence analysis of the PCR products of the long control region (LCR), the E5 and E7 open reading frames (ORFs), a segment of the L2 ORF overlapping the early viral poly(A) signal and a small segment of the L1 ORF or clinical isolates from Barbados and The Netherlands. Despite the widely different geographical and ethnic origin of the two groups of specimens, sequence analysis revealed relatively few mutational differences. Analysis of the LCR and the E5 ORF appeared to be the minimum requirement for the correct positioning of these variants in the HPV-16 phylogenetic tree. Most of the Barbadian variants appeared to be located at a unique position in the HPV-16 phylogenetic tree, at the internal branch close to the point where the European and Asian branches diverge. In contrast, most of the Dutch samples were located on the European branch.

Asia↗
PubMed1997

Marek's disease virus latency-associated transcripts belong to a family of spliced RNAs that are antisense to the ICP4 homolog gene.

Marek's disease virus (MDV) latency-associated transcripts include at least two MDV small RNAs (MSRs) and a 10-kb RNA which map antisense to the ICP4 homolog gene and are relatively abundant in MDV-transformed lymphoblastoid cells. This report further describes the biological and structural properties of these RNAs. First, these RNAs were detected in primary lymphomas isolated from chickens infected with several oncogenic MDV strains. Second, the MSRs are nonpolyadenylated, whereas, the 10-kb RNA is predominantly polyadenylated. Third, MSRs localize to the nuclei of both lymphoblastoid cells and cytolytically infected chicken embryo fibroblasts. Fourth, the 3'-region splice junctions of the MSRs during latent and productive infection were determined by sequencing RNA-PCR products generated with primers that flank the 3' splice region. The MSRs contain at least three introns, the largest of which overlaps the ICP4 putative translational start site. Fifth, the 5' end of the MSRs initiates approximately 5 kb upstream from the main body of the RNA. The extreme 5' exon is approximately 251 nucleotides (nt) long and is joined to the main body of the transcript upon removal of a 4,852-nt intron. Finally, the 10-kb RNA lies entirely within the repeats flanking the unique short region of the genome. We believe that the MSRs and 10-kb RNA belong to a family of spliced RNAs that map antisense to the ICP4 gene and comprise a complex transcriptional unit expressed during MDV-induced T-cell transformation.

Animals↗
PubMed2000

Regulation of stanniocalcin in MDCK cells by hypertonicity and extracellular calcium.

Differential display RT-PCR cloning method was applied to poly(A)(+) RNA isolated from Madin-Darby canine kidney (MDCK) cells in isotonic or hypertonic medium. A differentially expressed 360-bp PCR fragment was isolated, subcloned, sequenced, and used to screen an MDCK cDNA library constructed in lambdaZapII. A composite sequence of two overlapping cDNA clones provided 1,053 bp of sequence that was 93% identical to human stanniocalcin and corresponded to the 3'-end of the mRNA. Although the fish homolog of this hormone inhibits calcium uptake by the gill and intestine, the function of mammalian stanniocalcin remains unknown. Stanniocalcin cDNA probe hybridizes to a 4.4-kb mRNA that is induced eightfold by hypertonicity, in a manner that is dependent on medium organic osmolytes. The mRNA induction correlates with increased total cellular content of the protein and its concomitant release to the medium, consistent with secretion for autocrine or paracrine activity. Furthermore, induction of the mRNA by hypertonicity is dependent on extracellular calcium and displays a threshold phenomenon. The data suggest that kidney stanniocalcin may have a role in the adaptation of kidney cells to osmotic stress, in a manner that is extracellular calcium dependent.

Animals↗
PubMed1998

Identification of multiple CYP19 genes encoding different cytochrome P450 aromatase isozymes in brain and ovary.

Evidence to date indicates that the gene encoding cytochrome P450 aromatase (P450arom) in humans is a single member of the CYPl9 family, but multiple CYPl9 loci and isoforms have been identified in pigs. Here we report the cloning and characterization of a second member of the CYP19 family in goldfish. A search for P450arom variants was prompted by studies showing that a full-length P450arom complementary DNA (cDNA) isolated from a goldfish brain cDNA library hybridizes with a high abundance 3 kb transcript in brain RNA but fails to detect a message in ovarian RNA. A stepwise PCR cloning strategy led to isolation of a 1.9-kb cDNA, which encodes a protein of 518 amino acids and has a predicted mol wt of 58.7K. The ovary-derived P450arom (-A) shares 68-72% sequence identity with ovarian aromatases of other fish species, but only 62% identity with the homologous brain-derived P450arom (-B). Amino acid differences are distributed throughout the two goldfish P450arom forms, but presumptive functional domains are highly conserved. Both P450aromA and -B are able to aromatize [3H]androgen to [3H]estrogen when expressed in nonsteroidogenic COS cells. Southern analysis and PCR-restriction analysis of genomic DNA using discriminating probes and primers indicates that a single locus encodes the brain-derived P450aromB (CYPl9B), whereas one or two different loci encode the ovarian form (CYPl9A). Northern blot analysis revealed two P450aromA messenger RNAs (1.9 >> 3.0 kb) in ovary. Simultaneous PCR amplification with A- and B-specific primer pairs confirms that P450aromA is the only form expressed in ovaries, but shows overlapping expression of the two genes in neural tissues. Whereas P450aromB messenger RNA predominates in brain (B/A, approximately 14:1), the ratios are reversed in retina (B/A, approximately 1:25). Further studies are required to resolve the evolutionary and functional implications of multiple CYPl9 genes and P450arom isozymes in goldfish, their differential expression in brain and ovary, and whether observations can be generalized to other vertebrates.

Amino Acid Sequence↗
PubMed2001

[Cloning of the sugar related biosynthesis gene cluster from Streptomyces tenebrarius H6].

Streptomyces tenebrarius H6 produces a complex of aminoglycoside antibiotics, such as apramycin, tobramycin and kanamycin B etc. To study the apramycin biosynthetic genes the genomic library from the Streptomyces tenebrarius H6 was established using E. coli/Streptomyces shuttle vector pKC505 by in vitro packing. The probability of finding a specific gene from the library composed of 3,000 colonies was over 99.9%. According to the highly conserved sequence of the genes involved in 6-deoxyhexose biosynthesis, primers were designed and 0.6 kb fragment homologous to strE gene was obtained by PCR. 30 positive clones were found from the genomic library of S. tenebrarius H6 with the 0.6 kb fragment as a probe. Overlapped regions were localized by Southern hybridization and putative sugar related biosynthetic gene cluster was mapped by restriction enzyme digestions. An ORF of dTDP-glucose-4,6-dehydratase gene consisted of 1,132 bp, designated as aprE, was obtained and submitted to GenBank under the accession number of AF306787. A DNA sequence highly homologous to strL coding dTDP-4-dehydrorhamnose reductase was found linked with aprE gene.

Anti-Bacterial Agents↗
PubMed1991

Direct detection of dystrophin gene rearrangements by analysis of dystrophin mRNA in peripheral blood lymphocytes.

Using 10 overlapping nested sets of primers and using peripheral blood lymphocyte (PBL) total RNA as template, we have developed a system, based on PCR, which allows the rapid production of double-stranded cDNA corresponding to the entire coding sequence of the dystrophin gene. The product can be visualized on native minigels by ethidium staining and directly sequenced after gel purification. We have used this system to analyze the structures of PBL dystrophin mRNA in 26 Duchenne, Becker, or intermediate muscular dystrophy patients who have gross rearrangements of the dystrophin gene. In each case, the effect that the genomic rearrangement has on the structure of the transcript--and, by inference, on the dystrophin protein--has been determined, and the results confirm the frameshift hypothesis. The study also identifies a series of alternatively spliced transcripts which are specific to the rearranged genotypes and which seem therefore to arise following the alteration in the context of the splice signal. The system has been used for unambiguous identification of carrier females. Furthermore, the rapid production of microgram quantities of dystrophin cDNA from a readily accessible tissue makes point-mutation screening a practical proposition.

Base Sequence↗
PubMed1996

Construction of a 600-kilobase cosmid clone contig and generation of a transcriptional map surrounding the lung cancer tumor suppressor gene (TSG) locus on human chromosome 3p21.3: progress toward the isolation of a lung cancer TSG.

The critical region on human chromosome 3p21.3 harboring a putative lung cancer tumor suppressor gene (TSG) was previously defined by allelotyping and recently refined by overlapping homozygous deletions. We report the construction of a 700-kb (cosmid and one P1 phage) clone contig covering the deletion overlap and its flanks. The minimal set of 23 cosmids comprises 600 kb and is extended by one P1 phage to 700 kb to cover the distal breakpoint of the overlap. The clone contig was extensively characterized by restriction and expression mapping to produce high resolution physical and transcription maps of the cloned region. Potential transcribed fragments were detected by hybridization with PCR-amplified cDNA libraries, direct cDNA selection "zoo" blotting, cDNA screening, and identification of 24 CpG islands. Thus far, 15 new genes represented by partial or full-length cDNAs were isolated, characterized, and precisely positioned on the contig. Two previously cloned genes, namely GNAI-2 and GNAT-1, were also positioned. In addition, the telomeric breakpoint of the NCI H740 deletion and centromeric breakpoint of the overlapping GLC20 deletion were discovered and mapped to define precisely the candidate TSG region. This large cosmid clone contig and high resolution maps will prove crucial in the identification of the lung cancer TSG(s).

Chromosome Mapping↗
PubMed1997

Applications of the long and accurate polymerase chain reaction method in yeast molecular biology: direct sequencing of the amplified DNA and its introduction into yeast.

A DNA fragment longer than 10 kb can be amplified by the long and accurate polymerase chain reaction (LA-PCR) method. We demonstrate here applications of this technique in molecular biological studies of Saccharomyces cerevisiae. We have shown that DNA fragments amplified by LA-PCR can be directly used as a template in the chain-termination sequencing protocol, making it possible to quickly identify the DNA insert of yeast genomic library clones. We have also shown that the amplified yeast DNA can easily be introduced into yeast by co-transformation with linearized vector DNA. Overlapping DNA between the amplified yeast fragment and the vector must be more than 20 bp long in order to obtain 90% or more correct recombinant plasmids. These results suggest that simple amplification of yeast clones by LA-PCR can replace the previous procedures of yeast clone recovery, consisting of transformation of Escherichia coli, propagation of plasmids in E. coli and preparation of plasmid DNA.

Base Sequence↗
PubMed2005

[Polymerase chain reaction-single strand conformation polymorphism analysis combined with nucleic acid hybridization to detect ofloxacin-resistant Mycobacterium tuberculosis mutation selected in vitro].

OBJECTIVE: To explore the feasibility of utilizing polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis combined with Southern blot to detect ofloxacin (OFLX)-resistant Mycobacterium tuberculosis, and figure out the boundary between the OFLX-susceptible and resistant Mycobacterium tuberculosis. METHODS: OFLX-resistant Mycobacterium tuberculosis bacilli were induced in vitro and their minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) were determined. The 320 bp segments of gyrA gene were amplified by PCR and their polymorphism was tested by SSCP from H(37)Rv, H(37)Ra and those induced OFLX-resistant strains. To prove the repeatability of PCR-SSCP combined with Southern blot, 36 clinical isolated OFLX-resistant strains were tested. RESULTS: All of the 85 OFLX-resistant mutations involved in this study were identified to have gyrA mutations. The results showed that there was an overlap if OFLX 10 microg/ml was used as a criteria for discriminating susceptible and resistant strains. Compared with the H(37)Ra, the similar single-stranded shift caused by the single-stranded conformation change was viewed in the 36 clinical isolated OFLX-resistant strains by PCR-SSCP combined with Southern hybridization. CONCLUSIONS: PCR-SSCP analysis combined with Southern blot can clearly distinguish OFLX-susceptible from OFLX-resistant strains. The results suggest that as a cut-off point between OFLX-susceptible and low-level OFLX-resistant to Mycobacterium tuberculosis, OFLX concentration of 10 microg/ml is considered to be a better one.

Antitubercular Agents↗
PubMed2000

A multi-site study for detection of the factor V (Leiden) mutation from genomic DNA using a homogeneous invader microtiter plate fluorescence resonance energy transfer (FRET) assay.

The goal of this multicenter study was to evaluate the second-generation Invader technology for detecting the factor V (Leiden) mutation directly from genomic DNA of different sample types. Invader assay results were compared with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or allele-specific PCR (AS-PCR) analysis. The Invader assay is a PCR-independent methodology that uses a microtiter plate format. In the assay, a specific upstream Invader oligonucleotide and a downstream probe hybridize in tandem to a complementary DNA template and form a partially overlapping structure. The Cleavase VIII enzyme recognizes and cuts this structure to release the 5' flap of the probe. This flap then serves as an Invader oligonucleotide to direct cleavage of a fluorescence resonance energy transfer (FRET) probe in a second invasive cleavage reaction. Cleavage of this FRET probe results in the generation of a fluorescent signal. The results of the Invader assay were 99.5% concordant with the PCR-based methods. Of the 372 samples tested once, only two gave discordant results (one from operator error and one from unknown causes), but were concordant on retesting. These results indicate that a simple microtiter plate-based Invader assay can reliably genotype clinical patient samples for the factor V (Leiden) point mutation directly from genomic DNA without prior target amplification.

Base Sequence↗
PubMed1992

Human glial fibrillary acidic protein (GFAP): molecular cloning of the complete cDNA sequence and chromosomal localization (chromosome 17) of the GFAP gene.

We isolated three glial fibrillary acidic protein (GFAP) cDNA clones from a glioma cell line, U-251 MG. One clone isolated from a U-251 MG cDNA library was long, but lacked both ends. Using poly(A)+ RNA and primers synthesized according to the sequence of this clone, we used the polymerase chain reaction-assisted rapid amplification of cDNA ends (PCR-RACE) method, which is a strategy to isolate cDNA ends, and obtained cDNA clones for the 5' and 3' ends. From the sequences of these overlapping clones, the complete nucleotide sequence of human GFAP cDNA was established. The start (ATG) and the stop (TGA) signals were seen at nucleotide positions 15 and 1311, respectively, and divided the entire sequence of 3027 bp into 14 bp of 5' non-coding, 1296 bp of coding and 1717 bp of 3' non-coding regions. Using cDNA probes made from both the coding and the 3' non-coding regions, Northern blot hybridization was performed with two different stringencies on RNAs from human and rodent brains and human GFAP-positive and -negative cells. It was shown that the 3' non-coding region probe was more specific for human GFAP than the coding region probe which was specific only under higher stringency conditions. This was also suggested by homology analysis of the sequence with those of various intermediate filament proteins. Based on these findings, we performed spot blot hybridization of sorted human chromosomes and Southern blot hybridization of PCR-amplified DNAs of a panel of hamster-human somatic cell hybrids and localized the human GFAP gene to chromosome 17.

Amino Acid Sequence↗