Showing papers by "Michael McClelland published in 1993"

A genetic linkage map of Saccharum spontaneum L. 'SES 208'.

Abstract: The arbitrarily primed polymerase chain reaction was used to detect single-dose polymorphisms that, in turn, were used to generate a linkage map of a polyploid relative of cultivated sugarcane, Saccharum spontaneum 'SES 208' (2n = 64). The mapping population was composed of 88 progeny from a cross between SES 208 and a diploidized haploid derived from SES 208 by anther culture, ADP 85-0068. This cross allowed direct analysis of meiosis in SES 208 and gametic segregation ratios to be observed. One hundred twenty-seven 10-mer oligonucleotide primers of arbitrary sequence were selected from a pool of 420 primers used to screen the mapping parents. Three hundred thirty-six of the 420 primers amplified 4,540 loci or 13.5 loci per primer. The selected 127 primers revealed 2,160 loci of which 279 were present in SES 208 and absent in ADP 85-0068 and easily scored. Two hundred and eight (74.6%) of these 279 polymorphisms were single-dose polymorphisms (i.e., they displayed 1:1 segregation, chi 2 at 98% confidence level). Linkage analysis (theta = 0.25, LOD = 9.0 for two-point analysis, then theta = 0.25, LOD = 6.0 for multipoint analysis) of single-dose polymorphisms placed them into 42 linkage groups containing at least 2 markers. These single-dose markers span 1,500 contiguous centimorgans (cM) with 32 markers remaining unlinked (15.4%). From this 208-marker map we estimated the genome size of SES 208 to be 2,500 cM. The map has a predicted coverage of 85.1% at 30 cM, meaning that any new marker placed has an 85.1% chance of being within 30 cM of an existing marker. Furthermore, we show that SES 208 behaves like an autopolyploid because (i) the ratio of single-dose markers to higher dose markers fit the assumption of auto-octaploidy and (ii) the absence of repulsion phase linkages. This is the first genetic map constructed directly on a polyploid species for which no diploid relatives are known.

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

Abstract: 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.

RNA fingerprinting using arbitrarily primed PCR identifies differentially regulated RNAs in mink lung (Mv1Lu) cells growth arrested by transforming growth factor beta 1

Abstract: RNA fingerprinting using arbitrarily primed PCR (RAP) samples an RNA population and allows the detection of differentially expressed genes between two or more populations. This method was applied to mink lung epithelial cells, which respond to treatment with transforming growth factor beta (TGF-beta) by undergoing cell cycle arrest at or near the G1/S-phase boundary. The steady-state abundances of approximately 200 RNAs were surveyed, a few of which displayed differential regulation in response to TGF-beta 1. Three products were isolated, cloned, and sequenced. One differentially regulated RNA corresponded to cyclin A, a gene known to be required for the progression of mammalian fibroblasts through S phase. Northern blot analysis confirmed that the cyclin A mRNA steady-state abundance decreased dramatically in response to a 24-hr TGF-beta 1 treatment and also in response to cell cycle arrest caused by contact inhibition. A second RAP product corresponded to a previously unknown 7.5-kb mRNA, the level of which decreased dramatically in response to TGF-beta 1 treatment. Unlike the cyclin A mRNA, the abundance of this transcript did not decrease in response to growth arrest induced by contact inhibition. A third RAP product corresponded to the mRNA for osteonectin, an extracellular matrix protein. The abundance of this mRNA increased at least 2-fold during TGF-beta 1 treatment. This observation is consistent with other reports of increases in extracellular matrix proteins during TGF-beta treatment. RAP should be able to identify many of the genes that change in steady-state expression during the cell cycle.

Physical map of the genome of Rhizobium meliloti 1021.

Abstract: A physical map of the genome of Rhizobium meliloti 1021 is presented. The physical sizes of the three replicons in this genome had previously been determined and are as follows: the chromosome, 3.4 Mb; pSym-b, 1.7 Mb; and pSym-a, 1.4 Mb. The physical maps for this GC-rich genome contain AT-rich restriction sites for SwaI (5'-TAAATTTA-3'), PacI (5'-TTAATTAA-3'), PmeI (5'-GTTTAAAC-3'), and, for pSym-b, SpeI (5'-ACTAGT-3'). In addition, the endonuclease I-CeuI cleaved the 23S rRNA genes in this genome, and perhaps in most eubacterial genomes. I-CeuI digestion and polymerase chain reaction amplification of rrn regions were used to determine that there are at least three rrn loci in R. meliloti, all of which are located on the chromosome. The orientation of the rrn loci was determined by Southern blotting with probes from rrn sequences located 5' and 3' to the I-CeuI site. The rrn loci are clustered in one part of the chromosome and are oriented so that transcription will occur away from a single point in the circle, as observed for the origin of replication in the Escherichia coli and Salmonella typhimurium chromosomes. Fifteen genes that had been tagged by Tn5 insertion were localized to fragments on the chromosome physical map by using the IS50 as a probe in Southern blots. In addition, glt and gap were placed on the physical map by using Southern hybridization with cloned genes. The fortuitous occurrence of SpecI site in Tn5-233 was used to physically map 10 genetically mapped Tn5-233 integrations on pSym-b and to anchor the physical map to the genetic map. Finally, we demonstrate the usefulness of the map by localizing a total of 12 previously unmapped transposon insertions in the genome. This is the first physical map of the genome of a multireplicon member of the family Rhizobiaceae as well as the first physical map of a Rhizobium chromosome.

Effect of site-specific methylation on restriction endonucleases and DNA modification methyltransferases.

Abstract: We present in Table I an updated list of the sensitivities of 298 restriction endonucleases and 20 DNA methyltransferases to sitespecific modification at 4-methylcytosine (\"HT), 5-methylcytosine C^C), 5-hydroxymethylcytosine OTM^), and 6-methyladenine (^A) (McC14), four modifications that are common in the DNA of prokaryotes, eukaryotes, and their viruses (Mc2,Mc5,Mc8,Mcll,Ne3,Ne4). In addition, new information is included on restriction endonuclease cleavage at sites modified with 5-hydroxymethyluracil ( ^ U ) . Knowledge of the sensitivity of restriction endonucleases to site-specific modification can be used to study cellular DNA methylation. Several restriction-modification enzymes share the same recognition sequence specificity, but have different sensitivities to site-specific methylation. Table II lists 32 known isoschizomer pairs and one isomethylator pair, along with the modified recognition sites at which they differ. The data presented here and an additional three other tables are available in printed form or as a text file on a 3.5\" Macintosh diskette. The extra tables include Table HI which is a list of over 205 characterized DNA methyltransferases. A detailed list of cloned restriction-modification genes has been made by Wilson (Wi4). Table IV lists the sensitivities of over 20 Type II DNA methyltransferases to \"^C, C, C, and \"^A modification. Most DNA methyltransferases are sensitive to non-canonical modifications within their recognition sequences (Bu9,MclO, Ne3,Po4), and this sensitivity often differs from that of their restriction endonuclease partners. Table V gives a list of restriction systems in this review alphabetized by recognition sequence. The data can be supplied as a Microsoft Word, Macwrite or MS-DOS file. Please contact Michael McClelland at CTBR, phone 619 535 5486, FAX 619 535 5472.

Value of molecular epidemiologic analysis in a nosocomial methicillin-resistant Staphylococcus aureus outbreak.

Abstract: Objective. —To evaluate two molecular epidemiologic methods used in the analysis of a nosocomial methicillin-resistantStaphylococcus aureus(MRSA) outbreak. Design. —Restriction endonuclease analysis of plasmid DNA (REAP) was used in the analysis of 45 MRSA isolates. After termination of the outbreak, isolates were retrospectively analyzed in a blind fashion using the newly described technique of arbitrarily primed polymerase chain reaction (AP-PCR). Molecular analyses were compared with epidemiologic and antimicrobial susceptibility data. Setting. —Tertiary care university hospital. Subjects. —Twenty-eight patients and 12 employees infected or colonized with MRSA during a 6-week period. Results. —A clonal relationship demonstrated among isolates from burn unit patients and staff was clearly distinguishable from MRSA isolates arising from other hospital wards. The combination of REAP and AP-PCR provided complementary information in several instances. Aggressive measures to isolate infected patients and eradicate colonization from patients and staff terminated the outbreak. Conclusions. —Although traditional epidemiologic methods retain their central role in modern hospital infection control, molecular epidemiologic analysis can significantly enhance the ability of infection control officers to analyze and terminate hospital epidemics. The combination of AP-PCR and REAP may prove to be a particularly informative means of tracking the nosocomial spread of microbial strains and their mobile genetic elements. (JAMA. 1993;270:1323-1328)

Intervening sequence with conserved open reading frame in eubacterial 23S rRNA genes

Abstract: An intervening sequence (IVS) occurred in the 23S rRNA genes (rrl) of some, but not all, strains of four species of the spirochete genus Leptospira and was absent from strains in three other species. The IVS varied in size from 485 to 759 base pairs and replaced bases 1224-1245 in both copies of rrl. The two ends of each IVS shared 22-35 bases of complementarity that could form a stable double helix. The presence of an IVS correlated with a cleaved mature 23S rRNA that probably results from removal of the IVS without religation. The 3' site of cleavage was mapped within the inverted repeat of the IVS. An open reading frame of 121-133 amino acids was conserved in the IVS in all four species, oriented so that the sense strand was in the rRNA transcript. When the open reading frames were compared between species, they predicted polypeptides that showed between 51% and 78% amino acid conservation and similar DNA sequence conservation, indicating selection for protein function.

Arbitrary primed PCR fingerprinting of RNA applied to mapping differentially expressed genes.

Abstract: Differential gene expression between various tissues and developmental stages or between cells in vitro under different growth conditions can be rapidly and efficiently compared using the RNA arbitrarily primed polymerase chain reaction (RAP) fingerprinting method (Welsh et al, 1992b; Liang and Pardee, 1992) In RAP, a primer of arbitrary sequence primes both first and second strand cDNA synthesis The mixture of products is then PCR amplified and resolved electrophoretically, yielding highly reproducible fingerprints that are tissue-specific or growth condition-specific Differences between fingerprints arise from differentially expressed genes, as verified by Northern blot analysis RAP can be performed on the RNA samples using various DNA primers Each two day experiment yields a sample of approximately twenty cDNA products per lane making the identification of differentially or developmentally regulated genes no longer rate limiting Those PCR products representing genes that are regulated can be cloned from the gel and sequenced Sequences can be compared to the DNA and protein sequence databases to identify homologs, motifs and members of gene families The clones can be placed on the genetic map as Expression Tagged Sites (ETS, Adams et al, 1991a)

Identification of differentially expressed RNA in human ovarian carcinoma cells by arbitrarily primed PCR fingerprinting of total RNAs

Abstract: Using arbitrarily primed PCR fingerprinting of RNA (RAP), we have analyzed RNAs prepared from two normal ovarian surface epithelial cell cultures, two normal mesothelial cell cultures, and eight independent ovarian carcinoma cell lines. Each arbitrarily chosen primer gave a unique fingerprint of about 30 cDNA products. Most of the cDNA products produced by any particular primer were shared between all cell lines. However, one primer detected a cDNA PCR product that was absent in all eight ovarian carcinoma cell lines but present in all normal cell cultures. We have cloned and sequenced the DNA fragment that distinguishes normal from ovarian carcinoma cell lines. The DNA sequence has one continuous open reading frame throughout which indicates that it may be from a translated gene. Furthermore, we confirmed the differential expression of the gene by Northern blot analysis. We also observed that the intensity of the band in the RNA fingerprint correlates with the expression level of the corresponding RNA. These results further demonstrate the ability of RAP to detect differentially expressed genes in a quantitative manner and demonstrated the application of RAP for the detection of differential gene expression during carcinogenesis.

Species of Borrelia distinguished by restriction site polymorphisms in 16S rRNA genes

Abstract: Three phyletic groups of Borrelia associated with Lyme disease, B. burgdorferi, B. garinii and group VS461 can be distinguished from each other and other species of Borrelia by BfaI restriction site polymorphisms in PCR amplified 16S rRNA genes. One strain isolated from an Ixodes pacificus tick in California that was previously unclassifiable was distinguishable from B. burgdorferi by an MnlI restriction site polymorphism.

Identification of neoplasms by detection of genetic insertions and deletions

Abstract: The detection of insertions and/or deletions in reiterated nucleotide sequences in tissues provides an identification of neoplastic changes that are associated with malignancy. The mutations are preferably detected by PCR based amplification of target sequences using selected primers, followed by standard analytic procedures. The detection of these mutations is useful as a diagnostic tool for cancer development and has direct application for cancer prognosis.

Construction of a Physical Map of the Genome of Salmonella typhimurium

Abstract: The genus Salmonella belongs to the large, medically important eubacterial family Enterobacteriaceae It is closely related to Escherichia coli (Ochman and Wilson, 1987; Riley and Sanderson, 1990), but in E coli most strains are harmless commensals of mammals, whereas the salmonellae are ubiquitous pathogens of both warm-blooded and cold-blooded vertebrates The salmonellae were classified originally by somatic and flagellar antigens into the many serotypes of the Kauffman-White scheme, then were later shown to be part of a large “geno-species” or reassociation group according to DNA reassociation studies (Crosa et al, 1972) More recently, they have been separated using multi-locus enzyme electrophoresis into a number of electrophoretic types (ETs), which constitute clones (Selander et al, 1991)

TRT1 polynucleotides, host cells and assays

Abstract: A rapid method for generating a set of discrete DNA amplification products characteristic of a genome as a “fingerprint” comprises the steps of: priming target nucleic acid of a genome or from a cellular RNA preparation with an single-stranded primer to form primed nucleic acid such that a substantial degree of internal-mismatching occurs between the primer and the target nucleic acid; amplifying the primed nucleic acid by performing at least one cycle of polymerase chain reaction amplification; and amplifying the product of step (2) by performing at least about 10 cycles of polymerase chain reaction amplification. The method is known as the arbitrarily primed polymerase chain reaction (AP-PCR) method and is suitable for the identification of bacterial species and strains, mammals and plants. The method of the present invention can identify species, cell types or tissues rapidly, and does not require knowledge of the nucleotide sequence or other molecular biology of the nucleic acids of the organisms to be identified. The polynucleotide sequence LF9.5m, associated with normal growth of ovary cells, and the polynucleotide TRT1, associated with arrested cell growth, are specifically provided.

Leptospira Genomes Are Modified at 5'-GTAC

Abstract: Genomic DNAs of 14 strains from seven species of the spirochete Leptospira were resistant to cleavage by the restriction endonuclease RsaI (59-GTAC). A modified base comigrating with m4C was detected by chromatography. Genomic DNAs from other spirochetes, Borrelia group VS461, and Serpulina strains were not resistant to RsaI digestion. Modification at 59-GTAm4C may occur in most or all strains of all species of Leptospira but not in all genera of spirochetes. Genus-wide DNA modification has rarely been observed in bacteria. Images