Set Of Oligonucleotide Probes Research Articles

Detection of genital human papillomavirus by single-tube nested PCR and type-specific oligonucleotide hybridization.

Cervical cancer is, on a global scale, the second most common form of cancer in women. Development of cervical carcinoma is strongly associated with infection by certain types of human papillomavirus (HPV). To facilitate the detection and molecular typing of HPV in clinical samples, nested-PCR amplification systems were developed for regions of the E1 and L1 genes. The nested amplifications were performed in a single reaction tube, and shifting between inner and outer primer pairs was achieved by a two-phase amplification with different annealing temperatures. This method eliminates cross-contamination between samples during transfer from the first to the second amplification step. A set of type-specific oligonucleotide probes were designed for the E1 system and used to distinguish 19 genital HPV types. The sensitivities of our amplification systems compare favorably with that for the L1 system on the basis of the MY09-MY11 primer pair (M.M. Manos, Y. Ting, D. K. Wright, A. J. Lewis, T. R. Broker, and S. M. Wolinsky, Cancer Cells 7:209-214, 1989) and our systems can be used on materials such as HPV-infected cell lines, cytobrush samples, cancer biopsies, and recent as well as archival Papanicolaou (Pap) smears. The high sensitivity coupled with the effective elimination of contamination in the transfer between the two amplification steps of the nested PCR makes these systems suitable for research as well as clinical analyses.

Effects on DRB1 typing of two sets of sequence specific oligonucleotide probes

Effects on DRB1 typing of two sets of sequence specific oligonucleotide probes

The segregation and expression of glutamate receptor subunits in cultured hippocampal neurons

The distribution and expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-selective glutamate receptor subunits (GluR1-4) were studied in cultured hippocampal neurons using antibodies generated against peptides corresponding to the C-termini of GluR1, GluR2/3 and GluR4, and with a set of oligonucleotide probes designed complementary to specific pan, flip and flop GluR1-4 messenger RNA sequences. GluR1-4 subunit proteins were localized in fixed hippocampal neurons (2 h to three weeks after plating) by immunocytochemistry with light and electron microscopy. At early stages in culture, moderate staining with antibodies to GluR1 and GluR2/3 and very light staining with antibody to GluR4 was observed in cell bodies and proximal portions of all neurites of some neurons. Upon establishment of identified axons and dendrites by seven days in culture, staining was intense with specific antibodies to GluR1 and GluR2/3 and light with anti-GluR4 antibody in cell bodies and dendrites. Little or no staining was observed in axons. Cells at seven days in culture exhibited a variety of morphologies. However, we could not assign a pattern of staining to a particular type. As the cultures matured over two and three weeks, staining was limited to the somatodendritic compartment. The intensity of glutamate receptor subunit staining increased and the extent of staining proceeded to the distal extreme of many dendrites. Moreover, antibodies to GluR1-4 subunits were co-localized in neurons. Immunocytochemistry on living neurons did not result in any significant labeling, suggesting that the epitope is either not expressed on the surface of the neurons, or is present, but inaccessible to the antibody. Electron microscopy demonstrated receptor localization similar to that found in brain, with staining of postsynaptic membrane and density, dendritic cytoplasm and cell body, but not within the synaptic cleft. We examined the possible role of “cellular compartmentation” in the pattern of glutamate receptor expression in hippocampal neurons. Compartmentalization studies of the subcellular distribution of messenger RNAs encoding GluR1-4 subunits was determined in mature cultures by in situ hybridization. Significant silver grain appearance was restricted to the cell body, indicating that the synthesis of glutamate receptor subunits is limited largely to the neuronal cell body. The expression of microtubule-associated protein 2 was studied in parallel. Microtubule-associated protein 2 expression appeared 6 h after plating, while glutamate receptor subunit expression was present at 2 h. This indicates that microtubule-associated protein 2 does not regulate the initial distribution of glutamate receptor subunits into neurites. Restriction of expression to the cell body raises important questions concerning the mechanisms governing the transport of GluR proteins to their appropriate compartments within neurons of the developing and mature nervous system.

Cloning and nucleotide sequence of the gcv operon encoding the Escherichia coli glycine-cleavage system.

P-protein, H-protein and T-protein of the glycine cleavage system have been purified from Escherichia coli. Their N-terminal amino acid sequences were determined, and a set of oligonucleotide probes was designed for gene cloning. The nucleotide sequence of a fragment of DNA around the 62-min region of the E. coli chromosome, containing genes for the components of the glycine-cleavage system has been determined. The sequence includes three structural genes encoding T-protein (363 amino acids, 40013 Da), H-protein (128 amino acids, 13679 Da) and P-protein (956 amino acids, 104240 Da). These genes are named gcvT, gcvH and gcvP, respectively. They are organized in the above-mentioned order on the same strand of DNA with short intercistronic sequences. The presence of a potential promoter preceding gcvT and a typical rho-independent terminator sequence following gcvP indicated that the three genes constitute a single operon. Each component of the E. coli glycine-cleavage system exhibits considerable amino acid sequence similarity with the animal and plant counterparts. When the plasmid containing the gcv operon was transfected in E. coli cells, the gene products of gcvT, gcvH and gcvP were overexpressed under the direction of the promoter of the gcv operon. However, bacteria harboring the plasmid that contained the gcv operon without the promoter region and the 5' terminal portion of gcvT failed to overexpress any of the three components.

Sorbitol dehydrogenase from Bacillus subtilis. Purification, characterization, and gene cloning.

Cloning of the sorbitol dehydrogenase gene (gutB) from Bacillus subtilis offers an excellent system for studying zinc binding, substrate specificity, and catalytic mechanism of this enzyme through protein engineering. As a first step to clone gutB, B. subtilis sorbitol dehydrogenase has been purified to homogeneity and characterized. It is a tetrameric enzyme with a molecular mass of 38 kDa for each subunit. Atomic absorption analysis shows the presence of 1 mol of zinc atom/subunit. Substrate specificity and stereospecificity of the enzyme toward C-2 and C-4 of hexitols were established. Sequence of the first 31 amino acids was determined, and a set of oligonucleotide probes was designed for gene cloning. A positive clone carrying a 5-kilobase pair HindIII insert was isolated and sequenced. Sequence alignment indicated that the deduced amino acid sequence of B. subtilis sorbitol dehydrogenase shows 36% identity in sequence with the liver sorbitol dehydrogenase from sheep, rat, and human. In reference to the sequence of alcohol dehydrogenase, two potential zinc binding sites were identified. Sequence information related to the structure-function relationships of the enzyme is discussed.

Cloning and characterization of the groESL operon from Bacillus subtilis.

The sequence of the 10 N-terminal amino acids of a Bacillus subtilis protein that cross-reacts with antibody to Escherichia coli GroEL was used to design a set of degenerate oligonucleotide probes. These probes identified a clone which carries almost the entire groESL operon from a B. subtilis subgenomic library. By chromosomal walking, an additional fragment carrying the 3' end of groESL and its flanking sequence was isolated. Sequence analysis revealed two open reading frames (ORFs) in the cloned DNA. The upstream ORF encodes a 10-kDa protein which has 47% amino acid identity with E. coli GroES. The downstream ORF encodes a 58-kDa protein which is 62% identical to E. coli GroEL. A 2.1-kb groESL mRNA from B. subtilis was detected independently by Northern (RNA) blot analyses with a groES- and a groEL-specific probe. This demonstrated that groES and groEL are in an operon. The groESL promoter was located by using a promoter-probing plasmid, and the apparent transcription start site was mapped by primer extension analysis. The same promoter is utilized under normal and heat shock conditions. This promoter has the same features as a typical sigma A promoter. A strain in which the groESL operon was under the control of the sucrose-inducible sacB promoter was created. With this strain, it was possible to show that both groES and groEL are essential genes under both normal and heat shock conditions.

LINE-1 repetitive DNA probes for species-specific cloning from Mus spretus and Mus domesticus genomes

Mus domesticus and Mus spretus mice are closely related subspecies. For genetic investigations involving hybrid mice, we have developed a set of species-specific oligonucleotide probes based on the detection of LINE-1 sequence differences. LINE-1 is a repetitive DNA family whose many members are interspersed among the genes. In this study, library screening experiments were used to fully characterize the species specificity of four M. domesticus LINE-1 probes and three M. spretus LINE-1 probes. It was found that the nucleotide differences detected by the probes define large, species-specific subfamilies. We show that collaborative use of such probes can be employed to selectively detect thousands of species-specific library clones. Consequently, these probes could be exploited to monitor and access almost any given species-specific region of interest within hybrid genomes.

Cloning of a cDNA encoding 1-aminocyclopropane-1-carboxylate synthase and expression of its mRNA in ripening apple fruit

1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) purified from apple (Malus sylvestris Mill.) fruit was subjected to trypsin digestion. Following separation by reversed-phase high-pressure liquid chromatography, ten tryptic peptides were sequenced. Based on the sequences of three tryptic peptides, three sets of mixed oligonucleotide probes were synthesized and used to screen a plasmid cDNA library prepared from poly(A)(+) RNA of ripe apple fruit. A 1.5-kb (kilobase) cDNA clone which hybridized to all three probes were isolated. The clone contained an open reading frame of 1214 base pairs (bp) encoding a sequence of 404 amino acids. While the polyadenine tail at the 3'-end was intact, it lacked a portion of sequence at the 5'-end. Using the RNA-based polymerase chain reaction, an additional sequence of 148 bp was obtained at the 5'-end. Thus, 1362 bp were sequenced and they encode 454 amino acids. The deduced amino-acid sequence contained peptide sequences corresponding to all ten tryptic fragments, confirming the identity of the cDNA clone. Comparison of the deduced amino-acid sequence between ACC synthase from apple fruit and those from tomato (Lycopersicon esculentum Mill.) and winter squash (Cucurbita maxima Duch.) fruits demonstrated the presence of seven highly conserved regions, including the previously identified region for the active site. The size of the translation product of ACC-synthase mRNA was similar to that of the mature protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that apple ACC-synthase undergoes only minor, if any, post-translational proteolytic processing. Analysis of ACC-synthase mRNA by in-vitro translation-immunoprecipitation, and by Northern blotting indicates that the ACC-synthase mRNA was undetectable in unripe fruit, but was accumulated massively during the ripening proccess. These data demonstrate that the expression of the ACC-synthase gene is developmentally regulated.

Application of a rapid non-radioisotopic nucleic acid analysis system to the detection of sexually transmitted disease-causing organisms and their associated antimicrobial resistances.

We devised a versatile method for detecting nucleic acids in crude lysates of biological samples. A controlled network of nucleic acid hybrids composed of the target fragment, several oligonucleotide probes, branched DNA amplifiers, and labeled oligonucleotides is produced on a solid phase to ultimately incorporate 60 to 300 molecules of alkaline phosphatase, which are detected with a chemiluminescent substrate. The visible light output can be recorded on a luminometer or on instant black-and-white film. Assays have been developed for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and for genes conferring penicillin and tetracycline resistance. Conducted much like ELISAS, the assays are performed in about 4 h (for 96 samples) in microliter dishes. The molecular detection limit of approximately 50,000 molecules of double-stranded DNA has permitted us to detect 1 to 10 x 10(3) of C. trachomatis and N. gonorrhoeae with specific probe sequences. Both plasmid and genomic target sequences can be detected by the same procedure. All of the assay components, except for a set of unmodified oligonucleotide probes, are universally applicable for all targets.

Amino-acid substitutions at codon 13 of the N-ras oncogene in human acute myeloid leukaemia.

DNAs from four out of five patients with acute myeloid leukaemia (AML) tested by an in vivo selection assay in nude mice using transfected mouse NIH 3T3 cells were found to contain an activated N-ras oncogene. Using a set of synthetic oligonucleotide probes, we have detected a mutation at codon 13 in all four genes. The same codon is mutated in an additional AML DNA that is positive in the focus-formation assay on 3T3 cells. DNA from the peripheral blood of one patient in remission does not contain a codon 13 mutation.

Cloning the heavy chain of human HLA-DR antigen using synthetic oligodeoxyribonucleotides as hybridization probes.

The recent development of the amino acid microsequence technique allows us to obtain partial sequence information using an extremely small amount of protein. Two sets of mixed oligonucleotide probes were chemically synthesized using the amino acid sequence information for the heavy chain of human HLA-DR antigen obtained by the microsequence technique. These two hybridization probes were used to screen cDNA clones constructed from cytoplasmic poly(A)+ mRNA from a human B lymphoblastoid homozygous cell line (LG-2). Of the 10,000 clones screened, two clones hybridized with the probes. DNA sequence analysis showed that the longer one of the two cDNA clones was 1183 nucleotides long, including the entire coding region, the signal peptide region, and the complete 3'-noncoding region. The deduced amino acid sequence of the HLA-DR alpha chain is identical to that of other cell lines with a different HLA-DR typing. However, several nucleotide differences are found in the 3'-untranslated region compared with that of other DR haplotypes.