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M McClelland

Publications and source records attributed to M McClelland.

At least 91 records · Page 5Linked to original sources
PubMed1993

Leptospira species categorized by arbitrarily primed polymerase chain reaction (PCR) and by mapped restriction polymorphisms in PCR-amplified rRNA genes.

Reference strains from 48 selected serovars representing eight species of Leptospira were examined by two polymerase chain reaction (PCR)-based strategies. First, mapped restriction site polymorphisms (MRSP) were examined in PCR products from portions of rrs (16S rRNA gene) and rrl (23S rRNA gene). Twenty MRSP and 2 length polymorphisms were used to group reference strains into 16 MRSP profiles. Species assignments were consistent with those obtained by a second method, genomic fingerprinting with arbitrarily primed PCR, in which strains within a species were characterized by many shared arbitrarily primed PCR products. The results of both of these methods were in general agreement with those of previous studies that used DNA-DNA relatedness and confirmed the high level of divergence among the recognized species of Leptospira. However, Leptospira meyeri serovar ranarum and evansi strains were indistinguishable from some strains of Leptospira interrogans sensu stricto. Intervening sequences of about 485 to 740 bp were located near base 1230 in rrl of some strains.

Base Sequence↗
PubMed1993

The missing voice in interdisciplinary communication.

The research problem investigated in this article emerged from a year-long ethnographic study of the interdisciplinary process of teams evaluating children for mental retardation/developmental disabilities. The researchers noticed that when a discipline was not present at team meetings, others attempted to speak for it. This article examines the team process and case consequences of such a "missing voice." Data collected through participant observation and videotaping were triangulated with transcripts, written and audiovisual records, and an interview with the missing team member. This process revealed a trail of ambiguous findings and uncertainty on the part of team members of disciplines related to that of the missing member. The "missing" member identified the knowledge, assessment skills, and theoretical perspective she could have contributed if consulted. The findings demonstrate that (a) team members bring differing knowledge and observational perspectives and (b) team deliberations appear to be a function of who is present and what is negotiated.

Child↗
PubMed1992

Arbitrarily primed PCR fingerprinting of RNA.

Fingerprinting of RNA populations was achieved using an arbitrarily selected primer at low stringency for first and second strand cDNA synthesis. PCR amplification was then used to amplify the products. The method required only a few nanograms of total RNA and was unaffected by low levels of genomic double stranded DNA contamination. A reproducible pattern of ten to twenty clearly visible PCR products was obtained from any one tissue. Differences in PCR fingerprints were detected for RNAs from the same tissue isolated from different mouse strains and for RNAs from different tissues from the same mouse. The strain-specific differences revealed are probably due to sequence polymorphisms and should be useful for genetic mapping of genes. The tissue-specific differences revealed may be useful for studying differential gene expression. Examples of tissue-specific differences were cloned. Differential expression was confirmed for these products by Northern analysis and DNA sequencing uncovered two new tissue-specific messages. The method should be applicable to the detection of differences between RNA populations in a wide variety of situations.

Animals↗
PubMed1992

Nonspecific primer and PCR generated hybridization probes for physical ordering large restriction fragments in complex genome of S. aureus.

Pulsed field gel electrophoresis (PFGE) allows separation of large restriction fragments from bacterial genome. Restriction fragments obtained by digestion of Staphylococcus aureus DNA with rare cutting enzymes (Sma I, and Csp I) were separated by PFGE. To arrange the physical order of the fragments generated by digestion with one enzyme, probes were prepared by nonspecific priming and polymerase chain reaction (PCR), using individual fragments of the other enzymatic digest as a template. Probes were then used for Southern hybridization to the PFGE separated fragment distribution of the two infrequent cleaving enzymes (Sma I and Csp I). Using probes generated from four Sma I fragments and five Csp I fragments as individual templates, a partial physical order of Csp I fragments of the genome of S. aureus ISP8 has been determined in relation to a previously published Sma I map of S. aureus genome.

Base Sequence↗
PubMed1992

DNA mismatch correction by Very Short Patch repair may have altered the abundance of oligonucleotides in the E. coli genome.

A base mismatch correction process in E. coli K-12 called Very Short Patch (VSP) repair corrects T:G mismatches to C:G when found in certain sequence contexts. Two of the substrate mismatches (5'-CTWGG/3'-GGW'CC; W = A or T) occur in the context of cytosine methylation in DNA and reduce the mutagenic effects of 5-methylcytosine deamination to thymine. However, VSP repair is also known to repair T:G mismatches that are not expected to arise from 5-methylcytosine deamination (example--CTAG/GGT-C). In these cases, if the original base pair were a T:A, VSP repair would cause a T to C transition. We have carried out Markov chain analysis of an E. coli sequence database to determine if repair at the latter class of sites has altered the abundance of the relevant tetranucleotides. The results are consistent with the prediction that VSP repair would tend to deplete the genome of the 'T' containing sequences (example--CTAG), while enriching it for the corresponding 'C' containing sequences (CCAG). Further, they provide an explanation for the known scarcity of CTAG containing restriction enzyme sites among the genomes of enteric bacteria and identify VSP repair as a force in shaping the sequence composition of bacterial genomes.

5-Methylcytosine↗
PubMed1992

Biased DNA repair.

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Amino Acid Sequence↗
PubMed1992

Analysis of large DNA from soybean (Glycine max L. Merr.) by pulsed-field gel electrophoresis.

The technique of pulsed-field gel electrophoresis (PFE) has been used to study chromosomal regions and entire genomes of several organisms. Techniques are presented for the isolation of high molecular weight DNA from embedded soybean protoplasts and the conditions for separating large DNA fragments using PFE. Digestion was detected by Southern hybridization using single copy nodulin clones. These data are being used to generate a physical map of the nodulin region(s) of the soybean genome.

DNA↗
PubMed1992

Genomic fingerprinting by arbitrarily primed polymerase chain reaction resolves Borrelia burgdorferi into three distinct phyletic groups.

The causative agent of Lyme disease, Borrelia burgdorferi, was first identified by Burgdorfer et al. in 1982 (W. Burgdorfer, A. G. Barbour, S. F. Hayes, J. L. Benach, E. Grunwaldt, and J. P. Davis, Science 216:1317-1319, 1982) and was isolated by Barbour et al. in 1983 (A. G. Barbour, W. Burgdorfer, S. E. Hayes, O. Peter, and A. Aeschlimann, Curr. Microbiol. 8:123-126, 1983). Since then, a large number of isolates have been collected, and there have been questions regarding the relationships among the various strains. Using genomic fingerprinting by an arbitrarily primed polymerase chain reaction, we resolved into three groups a collection of Eurasian and North American isolates of spirochetes that are generally categorized as B. burgdorferi. Group I strains have been identified in both North America and Eurasia, while strains belonging to Borrelia groups II and III have been found only in Eurasia. These same three groups have also been delineated by Baranton et al. (G. Baranton, D. Postic, I. Saint Girons, P. Boerlin, J.-C. Piffaretti, M. Assous, and P. A. D. Grimont, Int. J. Syst. Bacteriol. 42:370-375, 1992) by independent methods. Two isolates are distinct from all of the other strains in our collection but are clearly members of the genus Borrelia.

Bacterial Typing Techniques↗
PubMed1992

PCR-amplified length polymorphisms in tRNA intergenic spacers for categorizing staphylococci.

The intergenic spacers between some adjacent tRNA genes were shown to be polymorphic in length when closely related Staphylococcus species were compared. A simple procedure was developed to detect and sequence these tRNA intergenic length polymorphisms (tRNA-ILPs). A comparison of homologous tRNA gene sequences flanking these ILPs in three Staphylococcus species was used to derive primers for high-stringency amplification of the ILPs by the polymerase chain reaction (PCR). The detection of tRNA-ILPs by PCR allowed the classification of virtually all strains from the five species of Staphylococcus that were examined. The procedure used to identify, sequence and derive primers for PCR detection of tRNA-ILPs in Staphylococcus should be applicable to many other genera of eubacteria. These primers could be used on uncultured material such as clinical samples.

Base Sequence↗
PubMed1992

Dissection of the Salmonella typhimurium genome by use of a Tn5 derivative carrying rare restriction sites.

A polylinker with rare restriction sites was introduced into a mini-Tn5 derivative. These sites include M.XbaI-DpnI (TCTAGATCTAGA), which is rare in most bacterial genomes, SwaI (ATTTAAAT) and PacI (TTAATTAA), which are rare in G+C-rich genomes, NotI (GCGGCCGC) and SfiI (GGCCN5GGCC), which are rare in A+T-rich genomes, and BlnI (CCTAGG), SpeI (ACTAGT), and XbaI (TCTAGA), which are rare in the genomes of many gram-negative bacteria. This Tn5(pfm) (pulsed-field mapping) transposon carries resistance to chloramphenicol and kanamycin to allow selection in a wide variety of background genomes. This Tn5(pfm) was integrated randomly into the Salmonella typhimurium and Serratia marcescens genomes. Integration of the new rare SwaI, PacI, BlnI, SpeI, and XbaI sites was assayed by restriction digestion and pulsed-field gel electrophoresis. Tn5(pfm) constructs could be valuable tools for pulsed-field mapping of gram-negative bacterial genomes by assisting in the production of physical maps and restriction fragment catalogs. For the first applications of a Tn5(pfm), we bisected five of the six largest BlnI fragments in the S. typhimurium genome, bisected the linearized 90-kb pSLT plasmid, and used Tn5(pfm) and Tn10 to trisect the largest BlnI fragment.

Base Sequence↗
PubMed1992

A BlnI restriction map of the Salmonella typhimurium LT2 genome.

BlnI or AvrII (5'-CCTAGG) sites are very rare in the Salmonella typhimurium LT2 genome. BlnI was used to construct a physical map which was correlated with the genetic map by using three methods. First, Tn10 carries BlnI sites, and the extra restriction sites produced by 34 genetically mapped Tn10 insertions were physically mapped by using pulsed-field gel electrophoresis. Second, six genetically mapped Mud-P22 prophage insertions were used to assign BlnI fragments. Integration of Mud-P22 introduces 30 kb of DNA that can easily be detected by a "shift up" in all but the largest BlnI fragments. Finally, induced Mud-P22 insertions package more than 100 kb of genomic DNA adjacent to one side of the insertion. Some of the smaller BlnI fragments were localized by hybridization to a dot blot array of 52 lysates from induced Mud-P22 insertions. Of the 10 BlnI sites mapped, 6 probably occur in or near the 16S rRNA genes at about 55, 71, 83, 86, 88.5, and 89.5 min. There is one BlnI site in the 90-kb pSLT plasmid. Two additional BlnI fragments of about 7 and 4 kb have not been localized. The size of the genome was estimated as 4.78 Mb (+/- 0.1 Mb) excluding pSLT but including prophages Fels-1 and Fels-2. One BlnI fragment that maps between 55 and 59 min showed a 40-kb reduction in size in a strain cured of the approximately 40-kb Fels-2 prophage.

DNA Transposable Elements↗
PubMed1992

Length polymorphisms in tRNA intergenic spacers detected by using the polymerase chain reaction can distinguish streptococcal strains and species.

Intergenic tRNA spacers from strains of streptococcal groups A, B, and G were amplified by using the polymerase chain reaction (PCR) at low stringency with consensus tRNA gene primers. Cloning and sequencing showed that many of the homologous intergenic spacers differed in length between species. The sequences of the tRNA genes that flank these polymorphic spacers were determined and used to synthesize fully complementary primers. With these primers at high stringency, PCR products which varied in lengths from 53 to 71 bp, depending on the species or strain, were obtained from streptococcal DNAs, even in the presence of a 1,000-fold mass excess of human DNA. PCR products, the lengths of which could also be used for classification, were obtained at high stringency from a few genera closely related to Streptococcus. No products were obtained from genomic DNAs from more distantly related genera. Production of species- or strain-specific tRNA intergenic length polymorphisms with primers that generate characteristic products from a variety of species within the same genus should be applicable to many organisms, including those that would otherwise be difficult to culture or identify.

Base Sequence↗
PubMed1991

Genomic fingerprinting using arbitrarily primed PCR and a matrix of pairwise combinations of primers.

Polymorphisms in genomic fingerprints generated by arbitrarily primed PCR (AP-PCR) can distinguish between slightly divergent strains of any organism. Single oligodeoxyribonucleotide (oligo) primers have been used to generate such fingerprints, with the same primer being present at the 5' end of both strands for every PCR product. We used three arbitrary oligos, individually and in pairs, to generate six different genomic fingerprints of the same mouse genomic DNAs. Fewer than half of the products in genomic fingerprints generated using the oligos in pairs were the same as those produced by AP-PCR using one of the three oligos alone. Thus, a few oligos could be used in a very large number of single and pairwise combinations, each producing a distinct AP-PCR fingerprint with the potential to identify new polymorphisms. For example, 50 oligos can be used in a matrix of pairwise combinations to produce 2,500 fingerprints, in which at least half the data can be expected to be unique to each pair. We demonstrate this principle by using two oligos, alone and together, to generate three sets of fingerprints and map thirteen polymorphisms in the C57BL/6J x DBA/2J set of recombinant inbred mice.

Animals↗
PubMed1991

PCR with 5-methyl-dCTP replacing dCTP.

When dCTP is replaced by methyl5-dCTP in the polymerase chain reaction some templates cannot be efficiently amplified by Taq polymerase or Vent polymerase using standard cycling parameters. However, this phenomenon can be overcome by increasing the temperature of the denaturation steps to 100 degrees C, or by adding dITP to destabilize the m5dC:dG base pairs. Once the block to amplification of m5dC-substituted DNA was overcome, methylated DNA from the 'superpolylinker' of the plasmid pSL 1180 was used as a substrate to check the methyl-sensitivity of a variety of restriction endonucleases. The m5dC-substituted DNAs should also be valuable substrates for defining the specificity of methyl-dependent endonucleases.

Base Sequence↗
PubMed1991

Enzymatic cleavage of a bacterial chromosome at a transposon-inserted rare site.

The sequential use of the methylase M.Xbal (5'.TCTAGm6A) and the methylation-dependent endonuclease Dpnl (5'-Gm6A decreases TC) results in cleavage at 5'.TCTAGA decreases TCTAGA. This recognition sequence was introduced into a transposon derived from the Mu bacteriophage and transposed into the genome of the bacterium Salmonella typhimurium. M.Xbal methylation was provided in vivo by a plasmid containing the M.Xbal gene and the S. typhimurium genome was cleaved to completion by Dpnl at one or more sites, depending on the number of transposon insertions. The resulting genomic fragments were resolved by pulsed-field electrophoresis. The potential use of single M.Xbal/Dpnl cleavage sites as reference positions to map rare restriction sites is discussed.

Base Sequence↗