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Biomedical subjects

R V Lebo

Publications and source records attributed to R V Lebo.

At least 73 records · Page 4Linked to original sources
PubMed1987

Evolutionary implications of the human aldolase-A, -B, -C, and -pseudogene chromosome locations.

The aldolase genes represent an ancient gene family with tissue-specific isozymic forms expressed only in vertebrates. The chromosomal locations of the aldolase genes provide insight into their tissue-specific and developmentally regulated expression and evolution. DNA probes for the human aldolase-A and -C genes and for an aldolase pseudogene were used to quantify and map the aldolase loci in the haploid human genome. Genomic hybridization of restriction fragments determined that all the aldolase genes exist in single copy in the haploid human genome. Spot-blot analysis of sorted chromosomes mapped human aldolase A to chromosome 16, aldolase C to chromosome 17, the pseudogene to chromosome 10; it previously had mapped the aldolase-B gene to chromosome 9. All loci are unlinked and located on to two pairs of morphologically similar chromosomes, a situation consistent with tetraploidization during isozymic and vertebrate evolution. Sequence comparisons of expressed and flanking regions support this conclusion. These locations on similar chromosome pairs correctly predicted that the aldolase pseudogene arose when sequences from the aldolase-A gene were inserted into the homologous aldolase location on chromosome 10.

Base Sequence↗
PubMed1986

Human prion protein cDNA: molecular cloning, chromosomal mapping, and biological implications.

A human complementary DNA whose protein product is considered to be the major component of scrapie-associated fibrils in Creutzfeldt-Jakob disease, kuru, and Gerstmann-Straussler syndrome has been identified and characterized. The extensive homology of this gene sequence to the hamster PrP 27- to 30-kilodalton prion protein complementary DNA clone, and its existence as a single copy in the human genome, leads to the conclusion that this is the human prion gene. This human prion gene has been mapped to human chromosome 20, negating a direct link between the prion protein and Down's syndrome or the amyloid of Alzheimer's disease.

Amino Acid Sequence↗
PubMed1986

Detecting abnormal human chromosome constitutions by dual laser flow cytogenetics.

Our custom dual laser chromosome sorter has been used to characterize and isolate metaphase human chromosomes rapidly for gene mapping purposes. Herein, we tested how well this system could detect unknown abnormal human chromosome constitutions. These results were compared to those of conventional cytogenetic analyses by banding and photomicrography. The sorter was used to analyze each cell line stained with two different stain pairs: DIPI-chromomycin and Hoechst-chromomycin. In 20 min, two histograms representing 2 X 10(5) chromosomes each were collected for each stain pair. A blind study of 11 samples by flow analysis demonstrated excellent concordance between the abnormal chromosomes detected and the diagnoses of Giemsa-banded karyotypes. Aneuploidy was identified by changes in the number of chromosomes in each histogram peak, while rearrangements such as deletions and translocations caused shifts in the histogram peak positions. The direction and distance of histogram peak shifts are directly related to alterations in chromosome size and banding pattern. We conclude that dual-laser flow analysis may provide a rapid approach to the screening and diagnosis of chromosome abnormalities.

Chromosome Aberrations↗
PubMed1986

Mapping of human autosomal phosphoglycerate kinase sequence to chromosome 19.

In order to map human PGK sequences, DNA was prepared from 55 human-mouse somatic cell lines. The DNA was digested to completion with HindIII and Southern filters prepared. These filters were hybridized at high stringency conditions to a human PGK cDNA. Mouse and human X-linked and autosomal bands were distinguished and, in addition to known X-linked sequences, two autosomal PGK sequences were mapped: a 1-kb band to chromosome 19 and a 5-kb band to chromosome 6. The PGK cDNA probe was also hybridized to flow-sorted chromosomes confirming the presence of PGK sequences on the X chromosome and chromosomes 6 and 19.

Animals↗
PubMed1986

Human erythropoietin gene: high level expression in stably transfected mammalian cells and chromosome localization.

The glycoprotein hormone erythropoietin plays a major role in regulating erythropoiesis and deficiencies of erythropoietin result in anemia. Detailed studies of the hormone and attempts at replacement therapy have been difficult due to the scarcity of purified material. We used a cloned human erythropoietin gene to develop stably transfected mammalian cell lines that secrete large amounts of the hormone with potent biological activity. These cell lines were produced by cotransfection of mammalian cells with a plasmid containing a selectable marker and plasmid constructions containing a cloned human erythropoietin gene inserted next to a strong promoter. The protein secreted by these cells stimulated the proliferation and differentiation of erythroid progenitor cells and, with increased selection, several of these cell lines secrete up to 80 mg of the protein per liter of supernatant. Hybridization analysis of DNA from human chromosomes isolated by high resolution dual laser sorting provides evidence that the gene for human erythropoietin is located on human chromosome 7.

Animals↗
PubMed1986

Chromosomal localization of the human apolipoprotein B gene and detection of homologous RNA in monkey intestine.

A cDNA clone of the human apolipoprotein B-100 was used as a hybridization probe to detect homologous sequences in both flow-sorted and in situ metaphase chromosomes. The results indicate that the gene encoding this protein is on the distal end of the short arm of chromosome 2 (2p23-2p24). RNA isolated from monkey small intestine contained sequences (6.5 and 18 kilobases) homologous to the cDNA of apolipoprotein B-100. These results are consistent with the hypothesis that one gene codes for both the intestinal (B-48) and the hepatic (B-100) forms.

Animals↗
PubMed1986

cDNA cloning of human plasminogen activator-inhibitor from endothelial cells.

Full-length cDNA for plasminogen activator inhibitor (PAI-1) was isolated from a human umbilical vein endothelial cell (HUVEC) lambda gt11 cDNA library. Three overlapping clones were identified by immunologic screening of 10(6) recombinant phage using a rabbit anti-human fibrosarcoma PAI-1 antiserum. The fusion proteins encoded by these three clones also react strongly with a monoclonal mouse anti-human fibrosarcoma PAI-1 antibody. By nucleotide sequence analysis, PAI-1 cDNA encodes a protein containing 402 amino acids with a predicted, nonglycosylated molecular mass of 45 kD. Identity of this material as authentic PAI-1 was confirmed by the presence of high level homology with the primary amino acid sequence of an internal peptide prepared from purified rat hepatoma PAI-1. The predicted amino acid sequence also reveals extensive homology with other members of the serine protease inhibitor gene family. Cultured HUVECs contain two PAI-1 mRNA species, both encoded by a single gene, differing by 1 kb in the 3' untranslated region. The PAI-1 gene is located on human chromosome 7.

Amino Acid Sequence↗
PubMed1986

Existence of glucose-6-phosphate dehydrogenase-like locus on chromosome 17.

Hybridization of DNA samples prepared from flow-sorted human chromosomes with a cDNA probe for the X-linked glucose-6-phosphate dehydrogenase (G6PD) suggested the existence of the G6PD-like locus on chromosome 17. Southern hybridization analysis of endonuclease-digested DNA samples from the human-mouse hybrid cell line with human chromosome 17, and from control human and mouse cells, proved that not only X chromosomes, but also chromosome 17, contain DNA sequences that are hybridizable with the G6PD cDNA probe. The G6PD-like locus on chromosome 17 could be a putative pseudogene or a functional gene for the fetal brain-specific G6PD isozyme or other protein.

Chromosome Mapping↗
PubMed1985

Localization of the gene encoding the human interleukin-2 receptor on chromosome 10.

The human interleukin-2 receptor is an inducible growth factor receptor present on the surface of activated T lymphocytes. The receptor is required for a normal T-cell immune response. High-resolution fluorescence-activated chromosome sorting and DNA spot-blot analysis with complementary DNA's for the interleukin-2 receptor indicated that the receptor gene was located on chromosome 9, 10, 11, or 12. In situ hybridization studies showed that the interleukin-2 receptor gene is on the short arm of chromosome 10, p14----15.

Cell Line↗
PubMed1985

Gene mapping of Malaysian alpha thalassemias with alpha and zeta globin gene probes.

Restriction enzyme analysis of the alpha and zeta globin genes was carried out in four cases of Hb Bart's hydrops fetalis, in three patients with Hb H disease without Hb CoSp, in three patients with Hb H disease with Hb CoSp, in 47 individuals with alpha thalassemia trait, and in 47 normal individuals. All four cases of Hb Bart's hydrops fetalis resulted from deletions of alpha 1 and alpha 2 globin genes which did not extend to the psi zeta 1 and zeta 2 globin genes. The same type of deletion was observed in alpha thal1 carriers, but two newborns (one Malay and one of Chinese extraction) had a nondeletion type of alpha thal1 which was confirmed by quantitative alpha globin gene analysis. In addition, two other newborns diagnosed as alpha thal1 trait carriers (one Malay, one Chinese) were shown to have a deletion of both alpha globin genes by quantitative alpha globin gene analysis, but further testing with zeta globin gene probe failed to reveal an abnormal fragment length characteristic of an alpha globin gene deletion. We believe that this last condition is due to a large deletion which includes all alpha globin genes and all zeta globin genes on the same chromosome. On another front, Bgl II restriction analysis of all four Hb Bart's hydrops fetalis cases and the alpha thal1 trait carriers showed a 10.5-kb Bgl II restriction fragment, in the hydrops fetalis as a single band, while in the carriers this 10.5-kb fragment was accompanied by the usual normal 12.5-kb and 11.3-kb fragments. We report that this 10.5-kb fragment, previously thought to be specific for the Southeast Asian alpha thal1 gene deletion, is also common in normal individuals. Nevertheless, digestion with other enzymes can clearly differentiate the alpha thal1 and normal genotypes. We distinguish the findings in the alpha thalassemias from the extensive DNA polymorphism in the region of the alpha and zeta globin genes.

Chromosome Deletion↗
PubMed1985

Spot-blot analysis of sorted chromosomes assigns a fructose intolerance disease locus to chromosome 9.

The aldolase B gene was mapped to chromosome 9 using a rapid gene mapping system. This system uses a dual-laser sorter to identify and separate metaphase human chromosomes stained with either DIPI-chromomycin or Hoechst-chromomycin. Chromosome panels were constructed from a normal cell line by sorting 22 chromosome fractions directly onto nitrocellulose filters. Twelve labeled gene probes hybridized to the sorted chromosomal DNA fractions predicted by previous chromosome assignments. Eighteen newly cloned genes have been mapped using the same protocol.

Carbohydrate Metabolism, Inborn Errors↗
PubMed1985

Mapping parathyroid hormone, beta-globin, insulin, and LDH-A genes within the human chromosome 11 short arm by spot blotting sorted chromosomes.

Rearranged human chromosomes carrying segments of chromosome 11 were separated from the normal chromosome 11 by high-resolution chromosome sorting. Sorted chromosomes were tested with parathyroid hormone, beta-globin, insulin, and LDH-A gene-specific probes to determine the genes carried by each chromosome segment. Based on the gene content and karyotypes of these abnormal chromosomes, the parathyroid hormone, beta-globin, insulin, and LDH-A genes and the unique restriction fragment ADJ-762 are all located on the terminal band of the short arm of human chromosome 11 (band 11p15), with LDH-A proximal to the other loci.

Animals↗
PubMed1985

Human ferritin light chain gene sequences mapped to several sorted chromosomes.

The iron storage ferritin light-chain gene exhibits multiple restriction enzyme fragments which have been mapped by analyzing sorted human chromosomes. A dual laser chromosome sorter was used to construct spot-blot filter panels representing 22 chromosome fractions. Hybridization of radiolabeled human ferritin-L gene probe to spot-blot panels revealed the ferritin-L gene on more than one chromosome. Miniaturized restriction enzyme analysis was used to map each of the ferritin-L restriction fragments uniquely to one of three chromosomes. This combination of sorted chromosome analyses provides a rapid method to map homologous DNA sequences located on more than one chromosome.

Base Sequence↗
PubMed1985

Gene encoding human growth hormone-releasing factor precursor: structure, sequence, and chromosomal assignment.

We have isolated and characterized overlapping clones from phage lambda and cosmid human genomic libraries that predict the entire structure of the gene encoding the precursor to human growth hormone-releasing factor. The gene includes five exons spanning 10 kilobase pairs of human genomic DNA. There appears to be a segregation of distinct functional regions of the GRF precursor and its mRNA into the five exons of the gene. The DNA sequences of all exons, intron/exon boundaries, and 5' and 3' flanking regions are presented. Dot-blot analysis of DNA from high resolution dual-laser-sorted human chromosomes indicates that the single-copy growth hormone-releasing factor gene is located on human chromosome 20.

Amino Acid Sequence↗
PubMed1985

Isolation of duplicated human c-src genes located on chromosomes 1 and 20.

The oncogene (v-src) of Rous sarcoma virus apparently arose by transduction of the chicken gene known as c-src(chicken). We isolated DNA fragments representative of two src-related loci from recombinant DNA bacteriophage libraries of the human genome. One of these loci, c-src1(human), appeared to direct the synthesis of a 5-kilobase polyadenylated RNA that presumably encodes pp60c-src(human). Probes specific for the other locus, c-src2(human), did not hybridize to polyadenylated RNA prepared from a variety of human cell lines. Partial nucleotide sequence determinations of the loci demonstrated that c-src1(human) is highly related to chicken c-src and that c-src2(human) is slightly more divergent. The sequences imply that the final two coding exons of each human locus are identical in length to those of chicken c-src and that the location of an amber stop codon is unchanged in all three loci. c-src1(human) has been mapped to chromosome 20, and the second locus is located on chromosome 1. We conclude that c-src1(human) is the analog of c-src(chicken) and that the duplicated locus, c-src2(human), may also be expressed.

Animals↗
PubMed1985

The thyroglobulin gene resides on chromosome 8 in man and on chromosome 7 in the rat.

Human chromosomes were separated by a dual laser FACS sorter and their DNA hybridized with a thyroglobulin gene probe. A strong hybridization signal was obtained with DNA from chromosome 8. A panel of mouse-rat cell hybrids was used to determine the chromosomal localization of the rat thyroglobulin gene by the Southern blotting method. Comparison of the cytogenetic data with the hybridization signals obtained with the rat thyroglobulin probe allowed assignment of this gene to rat chromosome 7. It is concluded that the synteny relationship between the thyroglobulin gene and the c-myc oncogene has been conserved in rat and man.

Animals↗
PubMed1984

High-resolution chromosome sorting and DNA spot-blot analysis assign McArdle's syndrome to chromosome 11.

A rapid gene-mapping system uses a high-resolution, dual-laser sorter to identify genes from separate human chromosomes prepared with a new stain combination. This system was used to sort 21 unique chromosome types onto nitrocellulose filter papers. Several labeled gene probes hybridized to the sorted chromosomal DNA types predicted by their previous chromosome assignments. The skeletal muscle glycogen phosphorylase gene was then mapped to a portion of chromosome 11 by spot blotting normal and translocated chromosomes.

Animals↗