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PubMed1989

Isolation of a cDNA encoding Aspergillus oryzae Taka-amylase A: evidence for multiple related genes.

Complementary and genomic DNAs encoding Aspergillus oryzae Taka-amylase A (Taa) were cloned and sequenced. The coding sequence of the cDNA comprised the signal peptide [21 amino acids (aa)] and mature Taa (478 aa). The deduced aa sequence agrees well with the published aa sequence, except for one insertion, one deletion and ten aa substitutions. These differences might be due to the difference in the strains used. Sequence comparison of the cDNA and genomic DNA indicates the presence of eight introns ranging in size from 55 to 86 bp. Southern-blot analysis showed the presence of at least two Taa genes, and the second gene (Taa-G2) was isolated. All the intron/exon junctions follow the 'GT-AG' rule, except for intron I of the first gene (Taa-G1). The 5'-noncoding region was well conserved among the genomic genes and contained sequences similar to 'CAAT' and 'TATA' boxes at nucleotides -121 and -31, counted from the transcription start point, respectively. The 3'-noncoding regions, however, differed significantly from each other. Taa-G2 contains a sequence identical to that of several independent cDNA clones, suggesting that it may be the major transcribed gene in A. oryzae.

Amino Acid Sequence↗
PubMed1989

Rapid identification of DNA fragments containing promoters for RNA polymerase II.

We describe a direct procedure for screening genomic recombinant DNA libraries or restriction fragments of cloned DNA regions for RNA polymerase II promoters. Cellular polyadenylated mRNA is chemically de-capped by beta-elimination reaction and enzymatically re-capped with [alpha-32P]GTP by vaccinia guanylyl transferase. Since this enzyme only accepts di- or triphosphorylated 5' termini as a substrate, the mRNAs are labeled exclusively at the first nucleotide, irrespective of whether the mRNA was intact or fragmented before in vitro capping. By using in vitro-capped mRNA as a hybridization probe, recombinant DNA molecules or restriction fragments that carry a cap site (and thus likely an RNA polymerase II promoter) can directly be identified. Here, we demonstrate the applicability of this procedure by the isolation and characterization of several genomic DNA clones containing RNA polymerase II promoter sequences, that are highly active in liver.

Animals↗
PubMed1990

Sequence and factor requirements for faithful in vitro transcription of human 7SL DNA.

We have analysed the transcription of a functional human 7SL gene by RNA polymerase III (RNAPIII) in S100 extracts in vitro. Accurate and efficient synthesis of 7S L RNA depends on the presence of (i) an upstream sequence and (ii) an internal promoter element located within the first 22 bp of the gene. These findings were substantiated by DNase I footprinting. Mutations of the internal promoter identified the doublet CG [nucleotide (nt) +15/+16] outside the A-box homologue (nt +5 to +14) as being essential for both proper promoter function in the in vitro transcription assay and competition in the template-exclusion assay. Fractionation of S100 extracts identified two fractions required in addition to RNAPIII for faithful transcription of the gene. Each of these two fractions gave rise to one of two footprints observed in DNase I protection experiments, indicating that at least two DNA-binding factors are involved.

Base Sequence↗
PubMed1992

Homeobox-containing genes in the newt are organized in clusters similar to other vertebrates.

In vertebrates, the majority of homeobox (HBox) genes are found in four clusters and this structural organization is believed to be of functional importance. Many HBox genes sustain their expression in the appendages of the adult newt. To further understand their regulation, the genomic loci of four newt HBox genes (two from the human HBox (HOX)-2 complex and two from the HOX-3 complex) were analysed and compared with homologous loci in other vertebrates. Notophthalmus viridescens HBox (NvHBox) genes were selected from a lambda EMBL3 library and analysed by restriction mapping and nucleotide (nt) sequencing. The nt sequences of the NvHBox genes have a very high degree of homology (more than 90%) with the human and mouse HBox genes, HOX-3.3, HOX-3.4, HOX-2.7 and HOX-2.8. The sequences flanking the HBox are also very homologous to their human and mouse counterparts. Moreover, the size of the DNA spacer separating NvHBox-3.3 from NvHBox-3.4, and NvHBox-2.7 from NvHBox-2.8 in the newt is similar in the homologous regions of the mouse and human, despite there being a C value ten times greater in the newt genome. Finally, three of these NvHBox genes are expressed in the limbs of the adult newt.

Amino Acid Sequence↗
PubMed1993

The glutamate dehydrogenase-encoding gene of the hyperthermophilic archaeon Pyrococcus furiosus: sequence, transcription and analysis of the deduced amino acid sequence.

Glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon, Pyrococcus woesei, has been isolated, characterized and found to be very similar if not identical to the recently purified GDH from P. furiosus. Using a polymerase chain reaction, based on the N-terminal amino acid sequences of GDH, the P. furiosus gdh gene was identified, cloned into Escherichia coli and sequenced. The transcription start point of gdh has been mapped 1 nucleotide upstream from the ribosome-binding site. Using antiserum raised against purified GDH, expression of gdh was observed in E. coli. The deduced primary sequence of the P. furiosus GDH has been compared to various bacterial, archaeal and eukaryal GDHs and showed a high degree of similarity (32-52%).

Amino Acid Sequence↗
PubMed1989

Oligonucleotide fingerprint analysis of the genomes of two variants of bluetongue virus serotype 2 isolated in the U.S.A.

The genome segments of two electrophoretically distinct variants of bluetongue virus (BTV) Serotype 2 (Ona A and Ona B) from the U.S.A. were analyzed by double-dimension gel electrophoresis of RNase T1 produced oligonucleotides. Segments 1, 4, 5, 6, 7 and 10 were examined individually after separation by SDS-PAGE; and Segments 2 and 3, and 8 and 9, which were difficult to resolve, were fingerprinted as pairs. The Ona A and Ona B strains appeared to be closely related since corresponding segments were comparable, sharing 53-89% of the large oligonucleotides counted. Since the strains with the Ona A electropherotype preceded Ona B infection in Florida, U.S.A. and since Ona A was indistinguishable from the early African isolate of Serotype 2, Ona B was thought to be a variant of an Ona A strain. These data tend to support the hypothesis that Ona B could have evolved from Ona A as the result of point mutations or genetic drift.

Animals↗
PubMed1990

A study of the predominant genotypes of bovid herpesvirus 1 found in the U.K.

The genotypic classification of 84 U.K. isolates of bovid herpesvirus 1 was determined by the restriction endonuclease technique. Preliminary studies with six enzymes (EcoRI, HindIII, HpaI, BamHI, PstI and BstEII) showed that the principal genotypic variants, including the live vaccine strains used in the U.K., could be identified from the digestion patterns with just two endonucleases: HindIII and HpaI. Isolates from the 1960s were all categorised as genotype 2b. From 1977 onwards, genotype 1 has predominated in mainland Britain, with occasional type 2b isolates. Type 2b viruses were isolated from both respiratory and genital disease cases. There was no evidence of genotypes 2a or 3 in the U.K.

Animals↗
PubMed1995

Statistical properties of DNA sequences.

We review evidence supporting the idea that the DNA sequence in genes containing non-coding regions is correlated, and that the correlation is remarkably long range--indeed, nucleotides thousands of base pairs distant are correlated. We do not find such a long-range correlation in the coding regions of the gene. We resolve the problem of the "non-stationarity" feature of the sequence of base pairs by applying a new algorithm called detrended fluctuation analysis (DFA). We address the claim of Voss that there is no difference in the statistical properties of coding and non-coding regions of DNA by systematically applying the DFA algorithm, as well as standard FFT analysis, to every DNA sequence (33301 coding and 29453 non-coding) in the entire GenBank database. Finally, we describe briefly some recent work showing that the non-coding sequences have certain statistical features in common with natural and artificial languages. Specifically, we adapt to DNA the Zipf approach to analyzing linguistic texts. These statistical properties of non-coding sequences support the possibility that non-coding regions of DNA may carry biological information.

Algorithms↗
PubMed1989

M13 Bioprints: non-isotopic detection of individual-specific human DNA fingerprints with biotinylated M13 bacteriophage.

M13 bacteriophage was labelled by primer extension with biotin-11-dUTP and was successfully used as probe to study human hypervariable minisatellites (DNA fingerprints) in Southern blots. The method, which we called M13 Bioprints, presents two advantages over conventional DNA fingerprinting. First, it is easily accessible, since it utilizes the widely available M13 phage as a probe. Second, it makes use of biotin, which is safer, simpler and more economical than isotopic labels.

Bacteriophages↗
PubMed1990

Isolation of the DNA minisatellite probe MZ 1.3 and its application to DNA 'fingerprinting' analysis.

A minisatellite probe, MZ 1.3, detecting hypervariable fragment patterns was isolated from a human genomic library. A repetitive sequence of 27 bp length was identified which is contained in the probe approx. 40 times. The MZ 1.3 repeat shows variable homology of 53-73% to the repetitive sequence of the protein III gene of the bacteriophage M13 genome. Polymorphic restriction fragment patterns were found with MZ 1.3 using the enzymes Hinf I, BstN I, Hae III, Mbo I, PstI/Pvu II, and Rsa I. An average of 18 polymorphic fragments was observed using Hinf I as enzyme. The band sharing frequency after Hinf I digestion among unrelated individuals was determined to be 23.8 +/- 7.2%. An example for the application of MZ 1.3 to paternity testing in an incest case is given. The probe can be used with radioactive or non-radioactive detection systems. An approach is presented to compare polymorphic fragment patterns from individuals obtained by independent gel runs on the basis of relative band positions (RBP) and calculated in a computerized analysis.

Base Sequence↗
PubMedDate not supplied

A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing.

A convenient and rapid method for preparing soluble extracts from the nuclei of as few as 3 x 10(7) mammalian cells (miniextract procedure) is described. By several criteria, miniextracts are comparable to nuclear extracts prepared from large numbers of cells by the conventional procedure. Miniextracts are able to support efficient transcription of a variety of class II promoters. In addition, DNase I footprinting and gel retardation assays can be performed directly in miniextracts, enabling the detection of sequence-specific DNA-binding proteins. Besides transcription, miniextracts efficiently carry out pre-mRNA splicing and allow formation and fractionation of previously characterized splicing complexes. The small-scale procedure enables simultaneous preparation of multiple extracts from a variety of cell types under different experimental conditions. Moreover, the use of small amounts of cells allows minimal expenditure of valuable or expensive materials such as radioactive compounds. Consequently, the procedure is highly advantageous for biochemical analysis of transcription and RNA processing in mammalian cells.

DNA-Binding Proteins↗
PubMed1990

Direct sequencing of PCR products using the Maxam-Gilbert method.

Direct sequencing of polymerase chain reaction (PCR) products by using the Maxam-Gilbert method is described. In this method, one of the primers is end labeled. Thus it is possible to sequence the reaction product directly following purification using this chemical method.

Base Sequence↗
PubMed1989

A high-resolution, fluorescence-based, semiautomated method for DNA fingerprinting.

We describe a fluorescence-based method for the automated analysis of DNA fragments on polyacrylamide gels. A single-stranded oligonucleotide primer (18-mer) with a fluorochrome covalently bound to its 5'-end is annealed to a synthetic oligonucleotide to create a double-stranded oligonucleotide linker with a 5'-overhang complementary to a restriction enzyme site. Cosmid or plasmid DNA is digested with the appropriate restriction enzyme and then ligated to the fluorochrome-labeled linker. The labeled restriction fragments are loaded on a denaturing polyacrylamide gel in a commercially available DNA sequencer. As the restriction fragments migrate through the gel, they intersect a laser beam which excites the fluorochrome-labeled fragment. Fluorescence emission data are captured on a computer in real time and analyzed after the completion of electrophoresis. Fragment length is nearly linearly related to migration time. This method offers very near single-base resolution up to 400 bases and the ability to quantitate fragment size up to 2000 bases. The fluorochrome-labeling chemistry relies on straightforward enzymatic reactions and can be performed in a single reaction tube. Because four different fluorochromes can be used, each of 16 lanes on the gel can be used to analyze four different digest reactions, one in each color. One of the fluorochromes can be used to label size standards in each lane, eliminating interlane variability and allowing more precise estimates of fragment size. We apply the method to the analysis of overlapping cosmids.

Animals↗
PubMed1989

Characterization of a nondeleterious L1 insertion in an intron of the human factor VIII gene and further evidence of open reading frames in functional L1 elements.

We have characterized an insertional event in IVS-10 of the factor VIII gene in a pedigree containing a hemophilia A patient (JH-25). The inserted DNA is a 5' truncated L1 element that is 681 bp long followed by a 3'66-bp poly(A) tract. The L1 element is inserted 154 bp 5' to the start of exon 11 and is flanked by a 13- to 17-bp target site duplication. The L1 insertion is present in four generations of the patient's family. The maternal grandfather who carries the insertion does not have hemophilia A, indicating that the insertion is not the cause of hemophilia A in the patient. We have sequenced this insertion and two previously reported de novo L1 insertions in the factor VIII gene in patients JH-27 (3785 bp) and JH-28 (2132 bp). The three nucleotide sequences differ by 0.2-0.8%. All three of these L1 insertions have open reading frames (ORFs) (1192, 642, and 157 aa) and the three derived amino acid sequences are 98-99% identical. The previously reported sequence similarity between L1 3' ORFs and the polymerase domain of reverse transcriptases is maintained in the ORFs of the JH-27 and JH-28 L1 insertions. The presence of open reading frames and the close sequence similarity of these recently inserted L1 elements provide indirect evidence for the existence of a set of functional L1 elements that encode one or more proteins necessary for their retrotransposition.

Base Sequence↗
PubMed1990

Automated DNA sequencing of the human HPRT locus.

The complete sequence of 57 kb of the human HPRT locus has been determined using automated fluorescent DNA sequencing. The strategy employed increasingly directed sequencing methods: A randomly generated M13 library was sequenced to generate contiguous overlapping sets of sequences (contigs). M13 clones at the ends of these contigs were further sequenced using M13 (universal and reverse) and custom oligonucleotide primers to order the contigs and to complete the sequencing project. The human HPRT sequence includes 1676 bp 5' and 15,238 bp 3' to exons 1 and 9, respectively. The sequence contains 49 representatives of the Alu repeat, along with several other types of repetitive sequences. The Alu sequences exhibit a biased orientation, with those sequences in the first half of the locus oriented in the minus direction relative to transcription of the gene (3'----5' = 77%, P less than 0.005) and those sequences in the latter half of the locus oriented randomly (5'----3' = 67%, P less than 0.5). The development and performance of the sequencing strategy and the features of the human HPRT gene are presented.

Amino Acid Sequence↗
PubMed1990

Development of an automated procedure for fluorescent DNA sequencing.

We describe here the development of a procedure for complete automation of the dideoxynucleotide DNA sequencing chemistry using fluorescent dye-labeled oligonucleotide primers. This procedure combines rapid preparation of template DNA using a modification of the polymerase chain reaction, automation of the DNA sequencing reactions using a robotic laboratory workstation, and subsequent analysis of the fluorescent-labeled reaction products on a commercial automated fluorescent sequencer. Using this procedure, we were able to produce sufficient quantities of template DNA directly from bacterial colonies or bacteriophage plaques, perform the DNA sequencing reactions on these templates, and load the reaction products on the fluorescent DNA sequencer in a single work day. This scheme for automation of the fluorescent DNA sequencing method allows the fluorescent sequencer to be run at its full capacity every day and eliminates much of the labor required to obtain a high level of data output. Currently, we are able to perform and analyze 16 fluorescent-labeled reactions every day, with an average output of over 7000 bp per sequencer run.

Automation↗
PubMed1991

Hypervariable minisatellite DNA sequences in the Indian peafowl Pavo cristatus.

We report here for the first time the large-scale isolation of hypervariable minisatellite DNA sequences from a non-human species, the Indian peafowl (Pavo cristatus). A size-selected genomic DNA fraction, rich in hypervariable minisatellites, was cloned into Charomid 9-36. This library was screened using two multilocus hypervariable probes, 33.6 and 33.15 and also, in a "probe-walking" approach, with five of the peafowl minisatellites initially isolated. Forty-eight positively hybridizing clones were characterized and found to originate from 30 different loci, 18 of which were polymorphic. Five of these variable minisatellite loci were studied further. They all showed Mendelian inheritance. The heterozygosities of these loci were relatively low (range 22-78%) in comparison with those of previously cloned human loci, as expected in view of inbreeding in our semicaptive study population. No new length allele mutations were observed in families and the mean mutation rate per locus is low (less than 0.004, 95% confidence maximum). These loci were also investigated by cross-species hybridization in related taxa. The ability of the probes to detect hypervariable sequences in other species within the same avian family was found to vary, from those probes that are species-specific to those that are apparently general to the family. We also illustrate the potential usefulness of these probes for paternity analysis in a study of sexual selection, and discuss the general application of specific hypervariable probes in behavioral and evolutionary studies.

Animals↗
PubMed1990

Use of DNA restriction endonuclease digest and ribosomal RNA gene probe patterns to fingerprint Helicobacter pylori and Helicobacter mustelae isolated from human and animal hosts.

Variation amongst strains of Helicobacter pylori and Helicobacter mustelae was examined by DNA restriction endonuclease digestion and rRNA gene patterns generated using a non-radioactive probe. Chromosomal DNA was extracted from 30 cultures of H. pylori from human, Rhesus monkey and pig gastric mucosa, and from three H. mustelae isolates from ferret gastric mucosa. DNA fingerprinting with Hae III and Hind III showed H. mustelae was relatively homogeneous but revealed genomic heterogeneity within H. pylori with at least 18 different DNA patterns identifiable amongst the 30 isolates. Five sets of strains other than duplicates with matching DNA fingerprints were identified within H. pylori. The Peruvian isolates were the largest identical set and comprised eight isolates from four different patients with five consecutive isolates from one patient. The Rhesus monkey strains were a relatively homogeneous set as were several Australian human isolates. The study demonstrates that rRNA gene restriction patterns provide a simple but highly discriminatory electrophoretic fingerprint for H. pylori with potential for use as a novel epidemiological marker in addition to total DNA digest analysis.

Animals↗