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1.  Mycobacterium Species Identification and Rifampin Resistance Testing with High-Density DNA Probe Arrays 
Species identification within the genus Mycobacterium and subsequent antibiotic susceptibility testing still rely on time-consuming, culture-based methods. Despite the recent development of DNA probes, which greatly reduce assay time, there is a need for a single platform assay capable of answering the multitude of diagnostic questions associated with this genus. We describe the use of a DNA probe array based on two sequence databases: one for the species identification of mycobacteria (82 unique 16S rRNA sequences corresponding to 54 phenotypical species) and the other for detecting Mycobacterium tuberculosis rifampin resistance (rpoB alleles). Species identification or rifampin resistance was determined by hybridizing fluorescently labeled, amplified genetic material generated from bacterial colonies to the array. Seventy mycobacterial isolates from 27 different species and 15 rifampin-resistant M. tuberculosis strains were tested. A total of 26 of 27 species were correctly identified as well as all of the rpoB mutants. This parallel testing format opens new perspectives in terms of patient management for bacterial diseases by allowing a number of genetic tests to be simultaneously run.
PMCID: PMC84166  PMID: 9854063
2.  Alternative splice acceptor utilization during human immunodeficiency virus type 1 infection of cultured cells. 
Journal of Virology  1990;64(9):4093-4098.
The utilization of alternative splice acceptors for excision of the 5' major intron of human immunodeficiency virus type 1 RNA was observed after infection in vitro. Specific splice events were monitored by a cDNA-polymerase chain reaction. These splice events shared a common splice donor but utilized several alternative splice acceptors. In addition to identifying the previously documented splice acceptors for tat and nef (S. K. Arya, C. Guo, S. F. Josephs, and F. Wong-Staal, Science 229:69-73, 1985), nucleotide sequence analysis of cDNA-polymerase chain reaction fragments also revealed the following: (i) two splice acceptors 15 and 9 nucleotides upstream from the rev start codon, which are utilized to create transcripts suitable for specific rev expression; and (ii) use of the splice acceptor previously attributed to nef to generate a singly spliced, env-encoding transcript. Hybridization signals representing the nef/env, tat, and rev splice events increased in intensity between 6 and 12 h after infection of CEM cells with the LAV-1BRU strain of human immunodeficiency virus type 1. In contrast, the signal for utilization of the nef/env splice acceptor for the singly spliced env transcript appeared first at 12 h and increased to maximum intensity by 24 h. The nef/env splice acceptor was dominant at all time points examined. We propose that this dominance ensures efficient downstream splicing proximal to the env initiation codon in singly spliced transcripts. However, early after infection, the dominance of the nef/env splice acceptor appears to divert primary transcripts away from tat- and rev-specific processing paths. The relative proportions of hybridization signals representing these alternative splice events remained constant throughout the viral replicative cycle. This result suggests that trans-acting factors that might influence splice choices are not induced during infection, but rather that cis-acting, sequence-specific splice preferences determine the relative efficiency of alternative acceptor utilization.
PMCID: PMC247871  PMID: 2384914
3.  Nucleic acid amplification in vitro: detection of sequences with low copy numbers and application to diagnosis of human immunodeficiency virus type 1 infection. 
Clinical Microbiology Reviews  1989;2(2):217-226.
The enzymatic amplification of specific nucleic acid sequences in vitro has revolutionized the use of nucleic acid hybridization assays for viral detection. With this method, the copy number of a pathogen-specific sequence is increased several orders of magnitude before detection is attempted. The sensitivity and specificity of detection are thus markedly improved. Mullis and Faloona devised the first method of sequence amplification in vitro, the polymerase chain reaction (K.B. Mullis and F.A. Faloona, Methods Enzymol. 155:355-350, 1987). By this method, synthetic oligonucleotide primers direct repeated, target-specific, deoxyribonucleic acid-synthetic reactions, resulting in an exponential increase in the amount of the specific target sequence. The application of sequence amplification to viral detection was initially performed with human immunodeficiency virus type 1 and human T-cell lymphoma virus type I. In principle, however, this approach can be applied to the detection of any deoxyribonucleic or ribonucleic acid virus; the only requirement is that sufficient nucleotide sequence data exist to allow the synthesis of target-specific oligonucleotide primers. The use of target amplification in vitro will permit a variety of studies of viral pathogenesis which have not been feasible because of the low copy number of the viral nucleic acids in infected material. This approach is particularly applicable to the study of human retroviral infections, which are chronic and persistent and are characterized by low titers of virus in tissues. In addition, target amplification in vitro will facilitate the development of new methods of sequence detection, which will be useful for rapid viral diagnosis in the clinical laboratory.
PMCID: PMC358112  PMID: 2650862
4.  Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris. 
Molecular and Cellular Biology  1985;5(5):1111-1121.
The oxidation of methanol follows a well-defined pathway and is similar for several methylotrophic yeasts. The use of methanol as the sole carbon source for the growth of Pichia pastoris stimulates the expression of a family of genes. Three methanol-responsive genes have been isolated; cDNA copies have been made from mRNAs of these genes, and the protein products from in vitro translations have been examined. The identification of alcohol oxidase as one of the cloned, methanol-regulated genes has been made by enzymatic, immunological, and sequence analyses. Methanol-regulated expression of each of these three isolated genes can be demonstrated to occur at the level of transcription. Finally, DNA subfragments of two of the methanol-responsive genomic clones from P. pastoris have been isolated and tentatively identified as containing the control regions involved in methanol regulation.
PMCID: PMC366829  PMID: 3889590
5.  ScrFI: a new sequence-specific endonuclease from Streptococcus cremoris. 
Nucleic Acids Research  1982;10(24):8171-8179.
A novel sequence-specific endonuclease has been isolated from Streptococcus cremoris F. ScrFI recognises the sequence: (formula; see text) and cleaves as indicated by the arrow ( ). It is the first enzyme to recognise this sequence and the first endonuclease reported from the lactic streptococci used in dairy fermentations.
PMCID: PMC327077  PMID: 6298711
6.  A semi-automated method for the reading of nucleic acid sequencing gels. 
Nucleic Acids Research  1982;10(1):103-114.
A collection of computer programs is described which permit automatic entering of nucleotide sequence data directly from an autoradiograph into a computer. This collection, called DIGITPAD, makes use of a digitizing tablet for the data entry and allows the rapid and accurate transfer of the sequence into the computer.
PMCID: PMC326118  PMID: 7063397
7.  Two new restriction endonucleases from Proteus vulgaris. 
Nucleic Acids Research  1981;9(18):4525-4536.
Two novel sequence-specific endonucleases have been isolated from Proteus vulgaris, ATCC 13315. PvuI recognizes the sequence: 5' C G A T decrease C G 3' 3' G C increase T A G C 5' and PvuII recognizes the sequence: 5' C A G decrease C T G 3' 3' G T C increase G A C 5' and cleave as indicated by the arrow (decrease). PvuI is an isoschizomer of XorII, RshI, and XniI. No enzyme with the specificity of PvuII has been described previously.
PMCID: PMC327455  PMID: 6272209
8.  Hybridization properties of immobilized nucleic acids. 
Nucleic Acids Research  1987;15(13):5373-5390.
The 5'-end attachment of oligonucleotides to dextran supports facilitates the study of the hybridization properties of an immobilized oligonucleotide system. The hybridization properties which were studied include: hybridization capacity and kinetics, hybridization-complex stability, and reagents influencing hybridization efficiency. Results of these experiments reveal that the hybridization efficiencies of support-bound oligonucleotides were 75-80% and 40-50% for single-stranded oligonucleotide targets and long double-stranded targets, respectively. These hybridization efficiencies are dependent upon prehybridizing the support-bound oligonucleotides with dextran sulfate. In addition, comparisons of the relative hybridization efficiencies of the support-bound oligonucleotide and nitrocellulose-based systems have been made which indicate a retention of 13-28% of target sequences on the filters and a detection efficiency of 8-20%.
PMCID: PMC305967  PMID: 3601675
9.  A computer assisted method for the determination of restriction enzyme recognifion sites. 
Nucleic Acids Research  1978;5(11):4105-4127.
A computer program has been developed which aids in the determination of restriction enzyme recognition sequences. This is achieved by cleaving DNAs of known sequence with a restriction endonuclease and comparing the fragmentation pattern with a computer-generated set of patterns. The feasibility of this approach has been tested using fragmentation patterns of 0X174 DNA produced by enzymes of both known and unknown specificity. Recognition sequences are predicted for two restriction endonucleases (BbvI and SfaNI) using this method. In addition, recognition sequences are predicted for two other new enzymes (PvuI and MstI) using another computer-assisted method.
PMCID: PMC342737  PMID: 724510
10.  Computer programs for the assembly of DNA sequences. 
Nucleic Acids Research  1979;7(2):529-545.
A collection of user-interactive computer programs is described which aid in the assembly of DNA sequences. This is achieved by searching for the positions of overlapping common nucleotide sequences within the blocks of sequence obtained as primary data. Such overlapping segments are then melded into one continuous string of nucleotides. Strategies for determining the accuracy of the sequence being analyzed and reducing the error rate resulting from the manual manipulation of sequence data are discussed. Sequences mapping from 97.3 to 100% of the Ad2 virus genome were used to demonstrate the performance of these programs.
PMCID: PMC328034  PMID: 493154
11.  Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. 
Nucleic Acids Research  1987;15(9):3859-3876.
Two DNA fragments containing putative control regions regulating the expression of the alcohol oxidase (AOX) and dihydroxy-acetone synthase (DAS) genes from the methylotrophic yeast Pichia pastoris were used in the construction of vectors for the expression of the Escherichia coli lacZ gene. These vectors were transformed into P. pastoris host cells and employed in experiments to measure the control mechanisms employed by each promoter in the production of beta-galactosidase fusion products. Results in P. pastoris suggest that the processes used to regulate the expression of these gene fusions involve both repression/derepression and induction mechanisms. Expression of the AOX-lacZ and DAS-lacZ fusions was examined in Saccharomyces cerevisiae as well. Interestingly, beta-galactosidase was expressed in a regulated manner in the heterologous host.
PMCID: PMC340787  PMID: 3108861
12.  Analysis of substrate specificity of the PaeR7 endonuclease: effect of base methylation on the kinetics of cleavage. 
Nucleic Acids Research  1990;18(17):5063-5068.
In murine cells expressing the PaeR7 endonuclease and methylase genes, the recognition sites (CTCGAG) of these enzymes can be methylated at the adenine residue by the PaeR7 methylase and at the internal cytosine by the mouse DNA methyltransferase. Using nonadecameric duplex deoxyoligonucleotide substrates, the specificity of the PaeR7 endonuclease for unmethylated, hemi-methylated, and fully methylated N6-methyladenine (m6A) and C5-methylcytosine (m5C) versions of these substrates has been studied. The Km, Kcat, and Ki values for these model substrates have been measured and suggest that fully or hemi-m6A-methylated PaeR7 sites in the murine genome are completely protected. However, the reactivity of fully or hemi-m5C-methylated PaeR7 sites is depressed 2900- and 100-fold respectively, compared to unmodified PaeR7 sites. The implications of the kinetic constants of the PaeR7 endonuclease for these methylated recognition sites as they occur in murine cells expressing this endonuclease gene are discussed.
PMCID: PMC332124  PMID: 2402435
13.  Introduction and expression of the bacterial PaeR7 restriction endonuclease gene in mouse cells containing the PaeR7 methylase. 
Nucleic Acids Research  1988;16(24):11489-11506.
To study the factors essential for a functional restriction system, the PaeR7 restriction-modification system has been introduced and expressed in murine cells. Transfer of this system was accomplished in two steps. First, cells containing sufficient PaeR7 methylase to completely methylate the mouse genome were constructed. In the second step, the mouse metallothionein promoter-regulated, endonuclease expression vector linked to the hygromycin B resistance selection marker was used to transfect the high methylase-expressing cells. Sixty percent of the clones isolated contained PaeR7 endonuclease enzymatic activity. Transfected cells expressing both methylase and endonuclease were incapable of blocking infection by DNA viruses, and possible explanations are discussed.
PMCID: PMC339060  PMID: 2850539
14.  Nucleotide sequence of the PaeR7 restriction/modification system and partial characterization of its protein products. 
Nucleic Acids Research  1985;13(23):8441-8461.
Bal31 deletion experiments on clones of the PaeR7 restriction-modification system from Pseudomonas aeruginosa demonstrate that it is arranged as an operon, with the methylase gene preceding the endonuclease gene. The DNA sequence of this operon agrees with in vitro transcription-translation assays which predict proteins of 532 amino acids, Mr = 59,260 daltons, and 246 amino acids, Mr = 27,280 daltons, coincident with the methylase and endonuclease genes, respectively. These predicted values coincide with the measured molecular weights of the purified, denatured PaeR7 endonuclease and methylase proteins. The first twenty amino acids from the amino-terminus of the purified endonuclease exactly match those predicted from the DNA sequence. Finally, potential regulatory mechanisms for the expression of phage restriction are described based on the properties of several PaeR7 subclones.
PMCID: PMC322144  PMID: 3001639
15.  The isolation and characterization of the Escherichia coli DNA adenine methylase (dam) gene. 
Nucleic Acids Research  1983;11(3):837-851.
The E. coli dam (DNA adenine methylase) enzyme is known to methylate the sequence GATC. A general method for cloning sequence-specific DNA methylase genes was used to isolate the dam gene on a 1.14 kb fragment, inserted in the plasmid vector pBR322. Subsequent restriction mapping and subcloning experiments established a set of approximate boundaries of the gene. The nucleotide sequence of the dam gene was determined, and analysis of that sequence revealed a unique open reading frame which corresponded in length to that necessary to code for a protein the size of dam. Amino acid composition derived from this sequence corresponds closely to the amino acid composition of the purified dam protein. Enzymatic and DNA:DNA hybridization methods were used to investigate the possible presence of dam genes in a variety of prokaryotic organisms.
PMCID: PMC325756  PMID: 6300769

Results 1-15 (15)