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PubMed1991

Hybrid Bacillus (1-3,1-4)-beta-glucanases: engineering thermostable enzymes by construction of hybrid genes.

Hybrid (1-3,1-4)-beta-glucanase genes were constructed by extension of overlapping segments of the (1-3,1-4)-beta-glucanase genes from Bacillus amyloliquefaciens and B. macerans generated by the polymerase chain reaction (PCR). Four hybrid genes were expressed in Escherichia coli cells. The mature hybrid enzymes contain a 16, 36, 78, or 152 amino acid N-terminal sequence derived from B. amyloliquefaciens (1-3,1-4)-beta-glucanase followed by a C-terminal segment derived from B. macerans (1-3,1-4)-beta-glucanase. Biochemical characterization of parental and hybrid enzymes shows a significant increase in thermostability of three of the hybrid enzymes when exposed to an acidic environment thus combining two important enzyme characteristics within the same molecule. At pH 4.1, 85%-95% of the initial activity was retained after 1 h at 65 degrees C in contrast to 5% and 0% for the parental enzymes from B. amyloliquefaciens and B. macerans. After 60 min incubation at 70 degrees C, pH 6.0, the parental enzymes retained 5% or less of the initial activity whilst one of the hybrids still exhibited 90% of the initial activity. Of the parental enzymes B. macerans (1-3,1-4)-beta-glucanase had the lower specific activity while the hybrid enzymes exhibited specific activities that were 1.5- to 3-fold higher. These experimental results demonstrate that exchange of homologous gene segments from different species may be a useful technique for obtaining new and improved versions of biologically active proteins.

Amino Acid Sequence↗
PubMed1991

Molecular and functional analysis of the virus- and interferon-inducible human MxA promoter.

The virus- and interferon-inducible human MxA (IFI-78k) gene is a homologue of the murine influenza resistance gene Mx1. Three overlapping human cosmid clones covering most of the gene including its promoter region were isolated. Sequencing the 5' MxA cDNA derived by RT-PCR (reverse transcriptase-polymerase chain reaction) confirmed the most 5' putative transcriptional start site. The MxA promoter does not contain a TATA or CCAAT box but has three Interferon Stimulated Response Element (ISRE) motifs. Strong induction with type I interferons was demonstrated with a fragment containing only two ISREs in human L132 cells. This induced expression was not adversely affected by 2-aminopurine. However, the promoter showed constitutive expression in transiently or stably transfected murine LM cells.

Animals↗
PubMed1994

Sequence variation in the gp135 gene of Maedi visna virus strain EV1.

We have used the PCR and HIRT DNA obtained from MVV-infected tissue culture cells as a template to generate a number of independently derived clones representing overlapping fragments of the gp135 region (env) of Maedi visna virus (MVV) strain EV1. Sequencing these clones revealed that homology between selected regions of gp135 ranged from 93.2% to 99.8%. Four hypervariable regions and one large highly conserved region have been identified. These data provide information on the variability of EV1 env, which extends and complements the data previously available on env variability between geographically distinct isolates of MVV.

Amino Acid Sequence↗
PubMed2006

Comparison of promoters controlling on the sunflower mitochondrial genome the transcription of two copies of the same native trnK gene reveals some differences in their structure.

Two copies of native trnK (UUU) gene are encoded on the sunflower mitochondrial DNA. They lie within two 12-kb direct repeats, presumably generated by a duplication event. During an investigation aimed at detecting DNA regions activating the trnK1 and trnK2 genes, three distinct promoters have been identified. Their locations were deduced using standard procedures (RT-PCR, RNA capping and 5'RACE) usually employed for the detection of transcription initiation sites (TISs). Promoters P3 and P2 control two independent partially overlapping transcription units containing the trnK2 and ccb206 genes, respectively. Promoter P1 has been mapped about 5200 bp upstream of the trnK1 gene which is part of a transcription unit also containing exons c, d and e of the nad2 gene, 5' to the tRNA gene. Most probably this promoter is not alone in controlling this transcription unit because this DNA region could be cotranscribed, at least partially, starting from other two promoters located upstream of the trnC and trnN genes, respectively. These genes have been previously mapped in a 5' region adjacent to the cluster containing nad2 exons c, d and e and the trnK1 gene. The comparative analysis of promoters P3 and P1 suggests that the difference between them could be related to the duplication event generating the second copy of trnK gene. The availability of a high number of new promoters belonging to dicot mitochondrial genomes makes possible to note some of their specific features.

Base Sequence↗
PubMed2005

Persistent Epstein-Barr viral reactivation in young African children with a history of severe Plasmodium falciparum malaria.

Epstein-Barr virus (EBV) and Plasmodium falciparum have overlapping distributions and are thought to have causal interactions, particularly with regard to the aetiology of endemic Burkitt's lymphoma. Using real-time PCR, we quantified and compared EBV DNA levels in the blood before and after antimalarial treatment of age- and gender-matched groups of Gabonese children who presented with either mild or severe P. falciparum malaria. Following treatment, the prevalence of EBV DNA declined in the mild malaria group but increased in the severe malaria group, and a significantly higher proportion of the latter had EBV DNA detectable in their blood when they were healthy and parasite free (67% vs. 39%; P=0.013). High EBV DNA loads were associated with more malaria attacks and with elevated plasma concentrations of both TNF-alpha and IL-12p40. Significantly more under 5 year olds had EBV DNA, highlighting the strong age dependence of the interaction between the two pathogens. These findings confirm that EBV is reactivated during acute P. falciparum malaria but, importantly, also reveal that: (i) EBV activity persists at a higher frequency in children with a history of severe malaria; and (ii) higher peripheral blood EBV DNA loads are associated with susceptibility to more frequent P. falciparum episodes and with altered cytokine activity.

Antimalarials↗
PubMed1995

Sequence of RNA2 of the Helicoverpa armigera stunt virus (Tetraviridae) and bacterial expression of its genes.

The complete nucleotide sequence of RNA2 of Helicoverpa armigera stunt virus (HaSV), a member of the Tetraviridae, was determined by characterization of cloned cDNA and PCR products and direct sequencing of genomic RNA. The capped, positive sense, single-stranded RNA is 2478 nucleotides in length and has two overlapping open reading frames (ORFs) likely to be cistrons which are situated between terminal non-coding regions of 282 and 168 bases, 5' and 3', respectively. Extensive secondary structure of the RNA strand is indicated, including a tRNA-like structure at the 3' terminus which is the first such structure discerned in an animal virus. The first ORF encodes a 17 kDa PEST protein (p17) of unknown function while the second ORF encodes the 71 kDa coat protein precursor (p71) that is cleaved at an Asn-Phe site into the 64 kDa and 7 kDa coat proteins. The precursor coat protein is 66% identical to that of another tetravirus, the Nudaurelia omega virus, with most of the difference residing in a 165 amino acid region located in the middle of the sequence. Despite the extensive similarity, no serological relationship was observed between the two viruses, suggesting that the dissimilar region is exposed on the capsid exterior. Expression in bacteria of the two RNA2 gene products shows they are likely to be expressed by a leaky scan-through mechanism. Bacterial expression of p71 did not produce virus-like particles while expression of p17 produced large arrays of mostly hollow, hexagonal tube-like structures.

Amino Acid Sequence↗
PubMed2000

Identification of a gene cluster for antibacterial polyketide-derived antibiotic biosynthesis in the nystatin producer Streptomyces noursei ATCC 11455.

Streptomyces noursei ATCC 11455 produces the antifungal polyene antibiotic nystatin containing the deoxysugar moiety mycosamine. Part of the deoxythymidyl diphosphate (TDP)-glucose dehydratase gene (gdhA) known to be involved in deoxysugar biosynthesis was amplified by PCR from genomic DNA of S. noursei ATCC 11455. A gene library for S. noursei was made and screened with the gdhA probe. Several overlapping phage clones covering about 30 kb of the S. noursei genome were physically mapped. A partial DNA sequencing analysis of this region resulted in the identification of several putative genes typical of macrolide antibiotic biosynthetic gene clusters. A gene-transfer system for 5. noursei has been established, and gene deletion or disruption experiments within the putative biosynthetic gene cluster were performed. All of the knock-out mutants retained the ability to produce nystatin, suggesting that the identified gene cluster is not involved in biosynthesis of this antibiotic. Culture extracts from the wild-type strain and three knock-out mutants were analysed by TLC followed by a bioassay against Micrococcus luteus. Two antibacterial compounds were found to be synthesized by the wild-type strain while only one was produced by the mutants. This provided evidence for the involvement of the identified gene cluster in the biosynthesis of a presumably novel antibacterial macrolide antibiotic in S. noursei.

Anti-Bacterial Agents↗
PubMed1994

Development and characterization of recA mutants of Campylobacter jejuni for inclusion in attenuated vaccines.

Isogenic recA mutants of Campylobacter jejuni have been constructed for evaluation of their usefulness in attenuated vaccines against this major worldwide cause of diarrhea. The recA+ gene of C. jejuni 81-176 was cloned by using degenerate primers to conserved regions of other RecA proteins in a PCR. The C. jejuni recA+ gene encodes a predicted protein with an M(r) of 37,012 with high sequence similarity to other RecA proteins. The termination codon of the recA+ gene overlaps with the initiation codon of another open reading frame which encodes a predicted protein which has > 50% identity with the N terminus of the Escherichia coli enolase protein. A kanamycin resistance gene was inserted into the cloned recA+ gene in E. coli and returned to C. jejuni VC83 by natural transformation, resulting in allelic replacement of the wild-type recA gene. The resulting VC83 recA mutant displayed increased sensitivity to UV light and a defect in generalized recombination as determined by natural transformation frequencies. The mutated recA gene was amplified from VC83 recA by PCR, and the product was used to transfer the mutation by natural transformation into C. jejuni 81-176 and 81-116, resulting in isogenic recA mutants with phenotypes similar to VC83 recA. After oral feeding, strain 81-176 recA colonized rabbits at levels comparable to wild-type 81-176 and was capable of eliciting the same degree of protection as wild-type 81-176 against subsequent homologous challenge in the RITARD (removable intestinal tie adult rabbit diarrhea) model.

Amino Acid Sequence↗
PubMed2004

A fluorescence in situ hybridization map of 6q deletions in acute lymphocytic leukemia: identification and analysis of a candidate tumor suppressor gene.

With the objective of identifying candidate tumor suppressor genes, we used fluorescence in situ hybridization to map leukemia-related deletions of the long arm of chromosome 6 (6q). Twenty of 24 deletions overlapped to define a 4.8-Mb region of minimal deletion between markers D6S1510 and D6S1692 within chromosome 6 band q16. Using reverse transcription-PCR, we found evidence of expression in hematopoietic cells for 3 of 15 genes in the region (GRIK2, C6orf111, and CCNC). Comparison between our own and published deletion data singled out GRIK2 as the gene most frequently affected by deletions of 6q in acute lymphocytic leukemia (ALL). Sequence analysis of GRIK2 in 14 ALL cases carrying heterozygous 6q deletions revealed a constitutional and paternally inherited C to G substitution in exon 6 encoding for an amino acid change in one patient. The substitution was absent among 232 normal alleles tested, leaving open the possibility that heterozygous carriers of such mutations may be susceptible to ALL. Although low in all normal hematopoietic tissues, quantitative reverse transcription-PCR showed higher baseline GRIK2 expression in thymus and T cells than other lineages. Among T-cell ALL patients, 6q deletion was associated with a statistically significant reduction in GRIK2 expression (P = 0.0001). By contrast, elevated GRIK2 expression was measured in the myelomonocytic line THP-1 and in one patient with common ALL. Finally, we detected significant levels of GRIK2 expression in prostate, kidney, trachea, and lung, raising the possibility that this gene may be protective against multiple tumor types.

Adolescent↗
PubMed2006

Genomic and expression analysis of the 3q25-q26 amplification unit reveals TLOC1/SEC62 as a probable target gene in prostate cancer.

Gain at chromosome 3q25-q26 has been reported to commonly occur in prostate cancer. To map the 3q25-q26 amplification unit and to identify the candidate genes of amplification, we did fluorescence in situ hybridization and quantitative real-time PCR for gene copy number and mRNA expression measurements in prostate cancer cell lines and prostate cancer samples from radical prostatectomy specimens. The minimal overlapping region of DNA copy number gains in the cell lines could be narrowed down to 700 kb at 3q26.2. Of all positional and functional candidates in this region, the gene TLOC1/SEC62 revealed the highest frequency (50%) of copy number gains in the prostate cancer samples and was found to be up-regulated at the mRNA level in all samples analyzed. TLOC1/Sec62 protein was also shown to be overexpressed by Western blot analysis. Intriguingly, the TLOC1/SEC62 gene copy number was increased in prostate tumors from patients who had a lower risk of and a longer time to progression following radical prostatectomy. These findings make TLOC1/SEC62 the best candidate within the 3q amplification unit in prostate cancer. TLOC1/Sec62 protein is a component of the endoplasmic reticulum protein translocation machinery, whose function during prostate carcinogenesis remains to be determined.

Cell Line, Tumor↗
PubMed1998

Detailed deletion mapping on chromosome 1p32-p36 in human colorectal cancer: identification of three distinct regions of common allelic loss.

Recent studies have suggested the existence of one or several tumor-suppressor genes on chromosome arm 1p in colorectal tumors. To determine the localization of the putative tumor suppressor genes, we performed LOH analysis in 1p in colorectal tumors. A total of 48 paired normal and tumor DNAs of 46 colorectal tumor patients and 21 microsatellite markers on 1p32.1-p36.3 were used for PCR-LOH analysis. Three commonly deleted regions were found: i) 1p36.3 (10-cm); ii) 1p35.1-p36.3 (2-cm); and iii) 1p34.2-p35 (1-cm). These regions overlapped with those reported in several types of tumor. No significant associations were found between LOH and clinicopathologic features. The regions identified in the present study could harbor tumor suppressor genes that would also be associated with several types of human cancer.

Adult↗
PubMed2003

DeltaNp63alpha and TAp63alpha regulate transcription of genes with distinct biological functions in cancer and development.

The p63 gene shows remarkable structural similarity to the p53 and p73 genes. Because of two promoters, the p63 gene generates two types of protein isoforms, TAp63 and DeltaNp63. Each type yields three isotypes (alpha, beta, gamma) because of differential splicing of the p63 COOH terminus. The purpose of this study was to determine whether there is a functional link between the distinct p63 isotypes in their transcriptional regulation of downstream targets and their role in various cellular functions. TAp63alpha and DeltaNp63alpha adenovirus expression vectors were introduced into Saos2 cells for 4 and 24 h, and then gene profiling was performed using a DNA microarray chip analysis. Seventy-four genes (>2-fold change in expression) were identified that overlapped between two independent studies. Thirty-five genes were selected for direct expression testing of which 27 were confirmed by reverse transcription-PCR or Northern blot analysis. A survey of these genes shows that p63 can regulate a wide range of downstream gene targets with various cellular functions, including cell cycle control, stress, and signal transduction. Our study thus revealed p63 transcriptional regulation of many genes in cancer and development while often demonstrating opposing regulatory functions for TAp63alpha and DeltaNp63alpha.

Apoptosis↗
PubMed2006

[Antigenicity of hepatitis C virus F protein and serum prevalence of anti-F in HCV-infected patients].

OBJECTIVE: To examine the antigenicity of hepatitis C virus (HCV) F protein and investigate serum prevalence of anti-F in HCV-infected patients. METHODS: Eleven pairs of overlapping primers were used to synthesize the full-length HCV f gene, from which the truncated HCV f65 gene fragment was amplified by PCR. HCV f65 gene was then cloned into pET32a(+), and transformed into E. coli strain Plyss (DE3). This recombinant E.coli was induced by IPTG for the production of HCV F65 protein. The expressed HCV F65 protein, purified by Ni-NTA agarose, was further used in ELISA to detect serum anti-F, and to immunize rabbits for making polyclonal anti-F. The rabbit polyclonal anti-F was purified by Staphylococcus aureus protein A agarose. RESULTS: After recombinant pET32a(+)-f65 was constructed successfully, HCV F65 protein was expressed and purified. The purified HCV F65 protein was used as a capture antigen in ELISA to detect serum anti-F in HCV infected patients (n = 30). The result showed that the mean A450 value and the positive rate of serum anti-F were 0.125+/-0.061 and 63.3%, respectively. The rabbit-derived polyclonal anti-F reacted specifically with HCV F65 protein, of which the titer was 1:30,000. CONCLUSION: Our expressed HCV F65 protein is of antigenicity, and can be used to determine serum anti-F. Anti-F IgG does exist in the sera of the HCV-infected patients. Moreover, the rabbit-derived polyclonal anti-F can be used to detect HCV F protein.

Animals↗
PubMed2006

Molecular diversity of microbial community in acid mine drainages of Yunfu sulfide mine.

Two acid mine drainage (AMD) samples were studied by a PCR-based cloning approach, which were from Yunfu sulfide mine in Guangdong province, China. A total of 15 operational taxonomic units (OTUs) were obtained from the two AMD samples. The percentage of overlapped OTUs in two AMD samples was 42.1%. Phylogenetic analysis revealed that the bacterium in the two samples fell into four putative divisions, which were Nitrospira, alpha-Proteobacteria, beta-Proteobacteria, and gamma-Proteobacteria four families. Organisms of genuses Acidithiobacillus and Gallionella, which were in gamma-Proteobacteria family and beta-Proteobacteria family, respectively, were dominant in two samples. The proportions of clones affiliated with Gallionella in each sample were 47.2% (G2) and 16.9% (G1). The result suggested that organisms of Gallionella were a very important composition in microbial communities of the two AMD samples we studied. In addition, the PCR amplification of archaeal 16S rDNA genes form these two AMD samples have been performed with two sets of archaea-specific primers, but no PCR product found.

Acids↗
PubMed2004

Viral RNAs detected in virions of porcine adenovirus type 3.

It has been demonstrated that cellular and viral RNAs were packaged in the virions of human cytomegalovirus (CMV) and herpes simplex virus 1 (HSV 1), members of the Herpesviridae family, both of which are enveloped double-stranded DNA viruses. Here, we provide evidence suggesting that RNAs are packaged in the virions of porcine adenovirus type 3 (PAdV-3), which is a member of the Adenoviridae family, a non-enveloped double-stranded DNA virus. The RNAs packaged in PAdV-3 virions were enriched in the size range of 300-1000 bases long. By reverse transcription (RT) of RNAs isolated from purified PAdV-3 virions, PCR amplification, and DNA sequence analysis of PCR products, we determined the identities of some viral RNAs contained in PAdV-3 virions. The results indicated that the RNAs representing transcripts from E1A, E1B, DNA binding protein (DBP), DNA polymerase (POL), E4 and some of the late genes including pIIIA, pIII, pV, Hexon, 33 K, and fiber were detected from purified PAdV-3 virions. In contrast, we could not detect the RNAs representing transcripts of precursor terminal protein (pTP), 52 kDa, pX, or 100-kDa protein genes in purified virions. Because the transcripts of pIX, IVa2, E3, protease, pVI, pVII, and pVIII overlap with those of other genes in PAdV-3, we could not definitely conclude that RNAs representing these transcripts were packaged in virions although the expected DNA fragments were produced by RT-PCR in the RNAs isolated from purified virions.

Adenovirus E1A Proteins↗
PubMed1995

Rat testicular carboxylesterase: cloning, cellular localization, and relationship to liver hydrolase A.

We recently purified from rat liver microsomes a carboxylesterase, designated hydrolase A, that catalyzes the hydrolysis of para-nitrophenylacetate with high affinity (Km approximately 25 microM) and is very sensitive to the inhibitory effects of phenylmethylsulfonyl fluoride (PMSF). Based on its catalytic properties, isoelectric point, and N-terminal amino acid sequence, hydrolase A corresponds to the pI 6.1 esterase cloned from a rat liver cDNA library by Robbi et al. (Biochem. J. 269, 451-458, 1990). A PMSF-sensitive esterase with high affinity toward para-nitrophenylacetate is also present in testicular microsomes at levels that slightly exceed those in liver microsomes. Antibody against purified hydrolase A recognizes a 57-kDa protein in both liver and testicular microsomes, suggesting that hydrolase A is expressed to a high degree in both tissues. To determine whether the testicular carboxylesterase is identical to hydrolase A, a rat testicular cDNA library was constructed and screened with antibody against hydrolase A. A 709-bp cDNA was isolated from immunopositive clones. Screening the same cDNA library by polymerase chain reaction (PCR) with one primer based on the sequence of the 709-bp cDNA and one primer based on the sequence of the adjoining lambda gt11 arm yielded a 1.1-kb cDNA that overlapped with the 709 bp-sequence. Together these two cDNA fragments spanned a 1792-bp sequence with an opening reading frame encoding 518 amino acids, which corresponds to approximately 95% of the C-terminal sequence of the liver pI 6.1 esterase (i.e., hydrolase A). Except for four nucleotide differences at positions 479, 855, 1335, and 1350, the sequence of the testicular cDNA was identical to the cDNA sequence of the liver pI 6.1 esterase reported by Robbi et al. None these changes results in an amino acid substitution. However, these four base substitutions were not observed when a cDNA encoding hydrolase A was isolated from a rat liver cDNA library by PCR. These results establish that the same carboxylesterase, namely, hydrolase A, is expressed in rat liver and testis. The levels of mRNA for hydrolase A in various rat tissues was estimated from Northern blots probed with the 709-bp cDNA isolated from the rat testicular cDNA library. A approximately 2-kb mRNA for hydrolase A was detected in liver, testis, lung, and prostate, which confirms the tissue distribution of hydrolase A based on catalytic activity and Western immunoblotting.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2001

Quantification of human cytomegalovirus DNA in bone marrow transplant recipients by real-time PCR.

A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA in peripheral blood leukocytes (PBLs) of bone marrow transplantation patients. Unlike other teams, we quantified CMV and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene using a plasmid containing both sequences as an external standard. Tenfold serial dilutions of this plasmid yielded overlapping standard curves that allowed the quantification of CMV and GAPDH gene copies in an efficient and accurate manner. Sequential blood samples (164 specimens) were collected from 16 patients. PBLs were tested by the pp65 antigenemia assay and quantitative CMV and GAPDH gene PCRs. CMV DNA was detected by PCR in 13 patients a mean of 15 days prior to the appearance of antigenemia. The administration of anti-CMV drugs led to a rapid decrease in the numbers of viral copies and positive nuclei. Real-time PCR assay results correlated with those of the CMV pp65 antigenemia assay (P < 0.00001). The TaqMan assay may be a useful tool for rapid quantification of CMV infection and for monitoring of CMV reactivation in bone marrow transplantation recipients.

Adolescent↗
PubMed2005

Large deletions within the first intron in VRN-1 are associated with spring growth habit in barley and wheat.

The broad adaptability of wheat and barley is in part attributable to their flexible growth habit, in that spring forms have recurrently evolved from the ancestral winter growth habit. In diploid wheat and barley growth habit is determined by allelic variation at the VRN-1 and/or VRN-2 loci, whereas in the polyploid wheat species it is determined primarily by allelic variation at VRN-1. Dominant Vrn-A1 alleles for spring growth habit are frequently associated with mutations in the promoter region in diploid wheat and in the A genome of common wheat. However, several dominant Vrn-A1, Vrn-B1, Vrn-D1 (common wheat) and Vrn-H1 (barley) alleles show no polymorphisms in the promoter region relative to their respective recessive alleles. In this study, we sequenced the complete VRN-1 gene from these accessions and found that all of them have large deletions within the first intron, which overlap in a 4-kb region. Furthermore, a 2.8-kb segment within the 4-kb region showed high sequence conservation among the different recessive alleles. PCR markers for these deletions showed that similar deletions were present in all the accessions with known Vrn-B1 and Vrn-D1 alleles, and in 51 hexaploid spring wheat accessions previously shown to have no polymorphisms in the VRN-A1 promoter region. Twenty-four tetraploid wheat accessions had a similar deletion in VRN-A1 intron 1. We hypothesize that the 2.8-kb conserved region includes regulatory elements important for the vernalization requirement. Epistatic interactions between VRN-H2 and the VRN-H1 allele with the intron 1 deletion suggest that the deleted region may include a recognition site for the flowering repression mediated by the product of the VRN-H2 gene of barley.

Alleles↗