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PubMed1997

A new restriction fragment length polymorphism from Cryptosporidium parvum identifies genetically heterogeneous parasite populations and genotypic changes following transmission from bovine to human hosts.

Length and restriction site polymorphism within a 2.8-kb threonine-rich open reading frame from Cryptosporidium parvum was identified and used to determine the genotypes of isolates from calves and humans. In agreement with observations of other genetic loci, all calf isolates were identical at this locus. In contrast, human isolates showed two profiles, one found exclusively in humans and one a superposition of both profiles, which were indicative of heterogeneous parasite populations. PCR fingerprints were consistent with a change in the genetic profile of C. parvum isolates following transmission from bovine to human hosts.

Amino Acid Sequence↗
PubMed1991

Cloning of human heparan sulfate proteoglycan core protein, assignment of the gene (HSPG2) to 1p36.1----p35 and identification of a BamHI restriction fragment length polymorphism.

We have isolated a cDNA coding for the core protein of the large basement membrane heparan sulfate proteoglycan (HSPG) from a human fibrosarcoma cell (HT1080) library. The library was screened with a mouse cDNA probe and one clone obtained, with a 1.5-kb insert, was isolated and sequenced. The sequence contained an open reading frame coding for 507 amino acid residues with a 84% identity to the corresponding mouse sequence. This amino acid sequence contained several cysteine-rich internal repeats similar to those found in component chains of laminin. The HSPG cDNA clone was used to assign the gene (HSPG2) to the p36.1----p35 region of chromosome 1 using both somatic cell hybrid and in situ hybridization. In the study of the polymorphisms of the locus, a BamHI restriction fragment length polymorphism was identified in the gene. This polymorphism displayed bands of 23 and 12 kb with allele frequencies of 76 and 24%, respectively.

Amino Acid Sequence↗
PubMed1992

Restriction fragment length polymorphism of polymerase chain reaction products from vaccine and wild-type varicella-zoster virus isolates.

The nucleotide changes that result in two restriction endonuclease polymorphisms that differentiate wild-type varicella-zoster virus (VZV) from the vaccine strain were determined. Oligonucleotide primers that flank these sites were used to amplify the intervening sequences with the polymerase chain reaction to identify VZV DNA in clinical isolates. Restriction enzyme digestion of the amplification products distinguished vaccine and wild-type genomes from one another. This study confirms the feasibility of amplifying VZV sequences so that they may be detected in clinical specimens and provides a molecular epidemiological approach to strain identification of VZV in vesicular lesions.

Base Sequence↗
PubMed2002

PCR-restriction fragment length polymorphism analysis of a diagnostic 452-base-pair DNA fragment discriminates between Cryptosporidium parvum and C. meleagridis and between C. parvum isolates of human and animal origin.

Genomic DNAs from human Cryptosporidium isolates previously typed by analysis of the 18S ribosomal DNA locus (Cryptosporidium parvum bovine genotype, C. parvum human genotype, Cryptosporidium meleagridis, and Cryptosporidium felis) were used to amplify the diagnostic fragment described by Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg., 45:688-694, 1991). The obtained 452-bp amplified fragments were sequenced and aligned with the homologous Cryptosporidium wrairi sequence. Polymorphism was exploited to develop a restriction fragment length polymorphism method able to discriminate Cryptosporidium species and C. parvum genotypes.

Animals↗
PubMedDate not supplied

Restriction fragment length polymorphism analysis of Cryptosporidium parvum isolates of bovine and human origin.

Cryptosporidium parvum oocysts isolated from different hosts and geographical areas were compared by restriction endonuclease analysis of repetitive DNA: Iowa (bovine), Florida (bovine), New York (bovine), Peru (human), Brazil (human), and Mexico (human). Southern blot hybridization analysis was performed using the restriction endonuclease enzyme Eco RI and the DNA probe pV47-2. The probe hybridized with 18 bands present in all the isolates. The Brazilian, Mexican, and Peruvian human isolates had an additional common band of 4.3 kbp that was absent in the bovine isolates. Two extra bands of 14 and 12 kbp were present in the Brazilian isolate whereas the Mexican isolate had an extra band of 14 kbp. When the Iowa and Peru C. parvum isolates were passed twice through calves, oocysts recovered from both passages showed identical banding patterns, suggesting that recombination of the repetitive sequences was not altered during sexual reproduction. The DNA digested with other restriction endonucleases were tested confirming differences between isolates. A genomic DNA library is currently being produced to better define isolate variation in C. parvum.

Animals↗
PubMed1995

HLA-Cw allele analysis by PCR-restriction fragment length polymorphism: study of known and additional alleles.

We describe a technique for HLA-Cw genotyping by digestion of PCR-amplified genes with restriction endonucleases. Locus-specific primers selectively amplified HLA-Cw sequences from exon 2 in a single PCR that avoided coamplification of other classical and nonclassical class I genes. Amplified DNAs were digested with selected enzymes. Sixty-three homozygous cell lines from International Histocompatibility Workshop X and 113 unrelated individual cells were genotypes for HLA-Cw and compared with serology. The present protocol can distinguish 23 alleles corresponding to the known HLA-Cw sequences. Genotyping of serologically undetectable alleles (HLA-Cw Blank) and of heterozygous cells was made possible by using this method. Six additional HLA-Cw alleles were identified by unusual restriction patterns and confirmed by sequencing; this observation suggests the presence of another family of allele-sharing clusters in the HLA-B locus. This PCR-restriction endonuclease method provides a simple and convenient approach for HLA-Cw DNA typing, allowing the definition of serologically undetectable alleles, and will contribute to the evaluation of the biological role of the HLA-C locus.

Alleles↗
PubMed1989

Restriction fragment length polymorphisms associated with growth hormone and prolactin genes in Holstein bulls: evidence for a novel growth hormone allele.

Sperm DNA isolated from sons of three extensively used US Holstein bulls was screened for differences associated with the primary gene structure of the bovine growth hormone (bGH) and prolactin (bPrl) genes. Southern blot analysis of DNA digested with 10 restriction enzymes revealed that offspring from two of the three bull families exhibited polymorphisms around the bGH and bPrl genes. Restriction fragment length polymorphisms (RFLPs) around the bGH gene were detected with five enzymes, whereas three enzymes revealed RFLPs around the bPrl gene. At least three structural differences were predicted around the bGH gene. The most common variant hybridization pattern appeared to involve an insertion/deletion located downstream of the conserved 3' EcoRI site. The presence of RFLPs in the genes coding for these pituitary hormones within a familial line may provide the basis for genetic markers associated with lactation and mammary development.

Alleles↗
PubMed1994

Restriction fragment length polymorphism of the human CYP2E1 (cytochrome P450IIE1) gene and susceptibility to lung cancer: possible relevance to low smoking exposure.

Polymorphic metabolism of certain chemical carcinogens may result in differences in susceptibility to cancers. Human CYP2E1 (cytochrome P450IIE1) is an enzyme involved in the metabolic activation of precarcinogens such as nitrosamines. We detected a restriction fragment length polymorphism (RFLP) of the human CYP2E1 gene for the restriction endonuclease Dra I. The distribution of this polymorphism was examined among lung cancer patients (n = 91), patients with cancer of the digestive tract (n = 45) and controls (n = 76). A significant difference in the distribution was observed between lung cancer patients and controls (chi 2 = 11.4 with 2 df; p < 0.005). On the other hand, there was no significant difference between patients between cancer of the digestive tract and controls (chi 2 = 4.87 with 2 df; NS). This finding suggests that the Dra I polymorphism of the CYP2E1 gene is associated with susceptibility to lung cancer. In addition, an association was found between the amount of lifelong smoking exposure and the distribution of the genotypes of the RFLP among lung cancer patients. The distribution pattern seemed deviated from that of controls especially in the population of low smoking exposure. Our Northern blot analysis data using RNA from human liver autopsy samples suggest that the Dra I polymorphism might be associated with the gene expression of CYP2E1 at mRNA level.

Aged↗
PubMed1985

Use of restriction fragment length polymorphisms to determine the clonal origin of human tumors.

A novel strategy to determine the clonal origin of human tumors has been devised. The strategy involves the use of a cloned polymorphic X-chromosomal gene and two restriction endonucleases. The first endonuclease distinguishes the paternal and maternal copies of the gene through a DNA polymorphism of restriction fragment length. The second endonuclease distinguishes active from inactive copies of this gene through changes in DNA methylation. As illustrations of this strategy, three human cancers were each shown to be monoclonal. The analysis described should have a wide variety of clinical and experimental applications.

Autoradiography↗