Showing papers by "Michael McClelland published in 1998"

Identification and sequence analysis of a 27-kilobase chromosomal fragment containing a Salmonella pathogenicity island located at 92 minutes on the chromosome map of Salmonella enterica serovar typhimurium LT2.

Abstract: Using a genomic approach, we have identified a new Salmonella pathogenicity island, SPI-4, which is the fourth Salmonella pathogenicity island to be identified. SPI-4 was located at 92 min on the chromosome map and was flanked by the ssb and soxSR loci. The DNA sequence covering the entire SPI-4 and both boundaries was determined. The size of SPI-4 was about 25 kb and it contains 18 putative open reading frames (ORFs). Three of these ORFs encode proteins that have significant homology with proteins involved in toxin secretion. Another five ORFs encode proteins that have significant homology with hypothetical proteins from Synechocystis sp. strain PCC6803 or Acinetobacter calcoaceticus. The rest of the ORFs encode novel proteins, one of which has five membrane-spanning domains. SPI-4 is likely to carry a type I secretion system involved in toxin secretion. Furthermore, a previously identified locus (ims98), which is required for intramacrophage survival, was also mapped within the SPI-4 region. These findings suggested that SPI-4 is needed for intramacrophage survival.

Overexpression of Lerk-5/Eplg5 messenger RNA: a novel marker for increased tumorigenicity and metastatic potential in human malignant melanomas.

Abstract: The Lerks, ligands of eph-related receptor tyrosine kinases, are a rapidly expanding family of genes thought to play an important role in the development and oncogenesis of various tissues. However, very little experimental evidence supports this hypothesis. Using RNA fingerprinting, we detected increased expression of Lerk-5 mRNA in human melanocytes as a response to the tumor-promoting drug 12-O-tetradecanoylphorbol-13-acetate, which suggests a possible role of the Lerks in melanoma tumorigenesis and progression. Therefore, we studied Lerk-5 mRNA expression in various melanoma cell lines and tissues of melanocytic tumors by semiquantitative reverse transcription-PCR. Modest expression of Lerk-5 mRNA was found in two melanoma cell lines derived from early primary tumors (WM35 and WM1645B); two metastatic cell lines tested showed a 3.9-fold increased transcript abundance when compared to the primary cell lines (RPMI-7951 and SK-Mel5). Progeny of a melanoma cell line with very low Lerk-5 mRNA abundance (WM35) showed a 5-fold increase in Lerk-5 mRNA expression when it was selected for higher tumorigenicity and multicytokine resistance by passaging in nude mice or repeated high-dose UVB irradiation. Consistent with these experimental data, we found high levels of Lerk-5 mRNA expression in advanced primary malignant melanomas and metastases (n = 22) but significantly lower or undetectable mRNA expression in benign melanocytic nevi (n = 9; P < 0.001). We conclude that increased Lerk-5 expression possibly reflects or induces an increased potential of growth, tumorigenicity, and metastatic abilities in human melanomas. This makes the yet to be elucidated eph-related receptor tyrosine kinase/Lerk signaling system a potential new source for molecular markers as well as a target for new therapies.

Non-stoichiometric reduced complexity probes for cDNA arrays

Abstract: A method is presented in which the reduced complexity and non-stoichiometric amplification intrinsic to RNA arbitrarily primed PCR fingerprinting (RAP-PCR) is used to advantage to generate probes for differential screening of cDNA arrays. RAP-PCR fingerprints were converted to probes for human cDNA clones arrayed as Escherichia coli colonies on nylon membranes. Each array contained 18 432 cDNA clones from the IMAGE consortium. Hybridization to approximately 1000 cDNA clones was detected using each RAP-PCR probe. Different RAP-PCR fingerprints gave hybridization patterns having very little overlap (<3%) with each other or with hybridization patterns from total cDNA probes. Consequently, repeated application of RAP-PCR probes allows a greater fraction of the message population to be screened on this type of array than can be achieved with a radiolabeled total cDNA probe. This method was applied to RNA from HaCaT keratinocytes treated with epidermal growth factor. Two RAP-PCR probes detected hybridization to 2000 clones, from which 22 candidate differentially expressed genes were observed. Differential expression was tested for 15 of these clones using RT-PCR and 13 were confirmed. The use of this cDNA array to analyze RAP-PCR fingerprints allowed for an increase in detection of 10-20-fold over the conventional denaturing polyacrylamide gel approach to RAP-PCR or differential display. Throughput is vastly improved by the reduction in cloning and sequencing afforded by the use of arrays. Also, repeated cloning and sequencing of the same gene or of genes already known to be regulated in the system of interest is minimized. The procedure we describe uses inexpensive arrays of plasmid clones spotted as E.coli colonies to detect differential expression, but these reduced complexity probes should also prove useful on arrays of PCR-amplified fragments and on oligonucleotide chips. Genesobserved in this manuscript: H11520, U35048, R48633, H28735, M13918, H12999, H05639, X79781, M31627, H23972, AB000712, R75916, U66894, AF067817.

Comparison of Sample Sequences of the Salmonella typhi Genome to the Sequence of the Complete Escherichia coli K-12 Genome

Abstract: Raw sequence data representing the majority of a bacterial genome can be obtained at a tiny fraction of the cost of a completed sequence. To demonstrate the utility of such a resource, 870 single-stranded M13 clones were sequenced from a shotgun library of the Salmonella typhi Ty2 genome. The sequence reads averaged over 400 bases and sampled the genome with an average spacing of once every 5,000 bases. A total of 339,243 bases of unique sequence was generated (approximately 7% representation). The sample of 870 sequences was compared to the complete Escherichia coli K-12 genome and to the rest of the GenBank database, which can also be considered a collection of sampled sequences. Despite the incomplete S. typhi data set, interesting categories could easily be discerned. Sixteen percent of the sequences determined from S. typhi had close homologs among known Salmonella sequences (P 1e-20), of which 155 sequences (18%) had no close similarities to any sequence in the database (P > 1e-5). Eight of the 277 sequences had similarities to genes in other strains of E. coli or plasmids, and six sequences showed evidence of novel phage lysogens or sequence remnants of phage integrations, including a member of the lambda family (P < 1e-15). Twenty-three sample sequences had a significantly closer similarity a sequence in the database from organisms other than the E. coli/Salmonella clade (which includes Shigella and Citrobacter). These sequences are new candidate lateral transfer events to the S. typhi lineage or deletions on the E. coli K-12 lineage. Eleven putative junctions of insertion/deletion events greater than 100 bp were observed in the sample, indicating that well over 150 such events may distinguish S. typhi from E. coli K-12. The need for automatic methods to more effectively exploit sample sequences is discussed.

GeneUp: A Program to Select Short PCR Primer Pairs that Occur in Multiple Members of Sequence Lists

Abstract: A computer program is presented that selects a small set of shor t primer pairs for PCR to sample all the sequences in a use r -specified list of mRNAs. Such primer pairs could be used to increase the prob ability of sampling mRNAs of particular interest in differential di s play and to generate simplified hybridization probes for DNA chip s or arrays. The program uses simulated PCR to find pairs of primer s that sample more than one sequence in the list. A small set of such primer pairs is selected that give maximal coverage of the sequence s in the list. Primer pairs are excluded that: (i )generate simulated PCR products of the same size from a number of sequences in th e list, (ii )can easily form primer dimers, (iii )are outside a specified range of G+C content or (iv )occur in another list of undesirable s e quences, such as rRNAs and Alu repeats. Five lists consisting of from 48 – 285 cDNA sequences were used to test the program. A smal l number of pairs of primers, 8–10 bases in length, were selected tha t fit the above criteria and that generate one or more simulated PCR products in all or most of the cDNAs in each list .

Selection of pcr primer pairs to amplify a group of nucleotide sequences

Abstract: The present invention provides a method of determining a set of primer pairs for amplifying a group of related nucleotide sequences. A method of the invention is performed by identifying a group of related nucleotide sequences; generating the set of primers that matches each of the related nucleotide sequences; determining for each systematic pairing of each primer which of the related nucleotide sequences are amplified; and selecting from the systematic pairings a subset which amplifies all of the related nucleotide sequences. The invention also provides a method of using a set of primer pairs, which amplify a group of related nucleotide sequences, to identify nucleotide sequences related to the original group of nucleotide sequences. The invention also provides a computer apparatus for carrying out the computer-executed steps of the method. The invention further provides a computer program product comprising a signal bearing media for carrying out the method.

Physical Mapping and Fingerprinting of Bacterial Genomes using Rare Cutting Restriction Enzymes

Abstract: Pulsed-field gel electrophoresis (PFGE) has allowed the separation of DNA fragments of five million base pairs or more, which is about the same size or greater than most bacterial genomes. When a bacterial genome is cut into a few discrete fragments, then PFGE is an ideal tool for the construction of physical maps (see also Chapters 25 and 26).

Estrogen-responsive RING finger mRNA induction in gastrointestinal carcinoma cells following bile acid treatment

Abstract: The underlying molecular mechanisms of the tumor-promoting activity of bile acids such as chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) and the protective effect of ursodeoxycholic acid (UDCA) remain largely unclear. Using RNA arbitrarily primed PCR (RAP-PCR) for differential display, we identified, cloned and sequenced differentially expressed transcripts after treating gastric carcinoma cells (St 23132) with the bile acids CDCA, DCA and UDCA. One of these transcripts was identified to be an estrogen-responsive RING finger protein (efp) mRNA. The differential expression of efp in gastric cancer cells was confirmed by low stringency RT-PCR. efp mRNA levels were induced 3-fold in gastric carcinoma cells after CDCA and DCA treatment, whereas no change in expression was detected after UDCA treatment. Finally, treatment of the colon carcinoma cell line HT 29 with DCA resulted in a 2- to 5-fold induction of efp mRNA levels whereas UDCA did not induce efp. As expected, efp expression was also increased after 24 h of estrogen treatment. In summary, a synergy or a common pathway of tumor enhancement of bile acids and estrogen via efp in gastrointestinal carcinogenesis can be envisioned.

Applications of DNA and RNA Fingerprinting by the Arbitrarily Primed Polymerase Chain Reaction

Abstract: The related methods Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) and Random Amplified Polymorphic DNA (RAPD) were developed independently by our group (Welsh and McClelland, 1990) and by Williams et al. (1990). These methods were based on the observation that PCR performed at relatively low stringency (in the case of AP-PCR) or with low selectivity primers at high stringency (in the case of RAPD) yield a reproducible collection of products that depend on the template and primer sequences. Arrayed on an agarose or acry1-amide gel, this collection of products can be viewed as a “fingerprint” or “bar code” for the DNA template. Because of their dependence on template sequence, AP-PCR and RAPD can be used as methods for sampling in sequence space; diverse applications can be imagined, many of which have been demonstrated in the literature.

Genome Evolution in the Salmonellae

Abstract: The genus Salmonella, composed of many species that occupy different habitats, diverged from Escherichia over 100 million years ago (see also Chapter 17). S. typhimurium LT2 has been studied genetically for many years. We have used four major methods to collect data from pulsed-field gel electrophoresis (see Chapters 24–26) on the structure of the genome of several species of Salmonella: 1) The endonuclease I-CeuI to determine an rrn operon skeleton; 2) Tn10s, which are inserted in specific genes for transduction into new species, and physical mapping of the genes; 3) Double digestion following excision of fragments in agarose; 4) Mud-P22s (see also Chapter 27) as linking probes.

Arbitrarily Primed PCR Methods for Studying Bacterial Diseases

Abstract: The most common application of the polymerase chain reaction (PCR) IS to exponenttally amplify a specific known and predictable sequence from a complex mixture of nucleic acids This chapter describes a technique that, in contrast, uses PCR with arbitrary primers, to generate a fingerprint of PCR products from complex mixtures of nucleic acids and identify sequence polymorphisms and other differences that distinguish them This robust and reproducible technique can amplify discrete sets of DNA fragments When genomic DNAs from different individuals are compared, these differences represent point mutations and variously sized deletions and insertions When different sources of RNA are examined from an isogenic source, differences in the particular set of DNA fragments amplified represent differentially expressed genes

Selection de paires d'amorces pcr destinees a l'amplification d'un groupe de sequences nucleotidiques

Abstract: La presente invention concerne une methode permettant de determiner un ensemble de paires d'amorces destinees a l'amplification d'un groupe de sequences nucleotidiques associees. La methode de l'invention consiste a identifier un groupe de sequences nucleotidiques associees; a produire l'ensemble d'amorces correspondant a chacune des sequences nucleotidiques associees; a determiner pour chaque appariement systematique de chacune des amorces quelles sont les sequences nucleotidiques associees amplifiees; et a selectionner a partir des appariements systematiques, un sous-ensemble amplifiant toutes les sequences nucleotidiques associees. L'invention concerne egalement une methode d'utilisation d'un ensemble de paires d'amorces, methode qui consiste a amplifier un groupe de sequences nucleotidiques associees pour identifier des sequences nucleotidiques associees au groupe de sequences nucleotidiques d'origine. L'invention concerne aussi un dispositif informatique permettant de realiser les differentes etapes, executees de maniere informatisee, de l'invention. L'invention concerne enfin un logiciel comprenant un milieu vecteur de signaux permettant de mettre en oeuvre la methode precitee.