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Biomedical subjects

W Salser

Publications and source records attributed to W Salser.

At least 19 recordsLinked to original sources
PubMed1991

Stable methylation patterns of MYC and other genes regulated during terminal myeloid differentiation.

Changes in DNA methylation are often associated with the modulation of gene expression. The DNA methylation patterns of six genes whose expression is regulated with terminal myeloid maturation were examined in HL-60 cells before and after their differentiation towards granulocytes. Methylation patterns were stable in five of these genes even with strong up- or down-regulation of mRNA levels. A somatic hybrid cell formed from HL-60 and B-lymphoid cells was studied for mRNA expression and DNA methylation of several of these genes. The pattern of methylation of the genes studied in the hybrid cells was a chimera of the parental patterns. These data suggest that changes in DNA methylation may not be necessary for the modulation of expression of many genes during terminal myeloid differentiation. Furthermore, somatic hybrid cell experiments suggest that specific DNA methylation patterns from both parental cells are inherited in a chimeric pattern by the hybrid and are independent of gene expression.

Blotting, Northern↗
PubMed1990

Further mapping of an ataxia-telangiectasia locus to the chromosome 11q23 region.

We recently mapped the gene for ataxia-telangiectasia group A (ATA) to chromosome 11q22-23 by linkage analysis, using the genetic markers THY1 and pYNB3.12 (D11S144). The most likely order was cent-AT-S144-THY1. The present paper describes further mapping of the AT locus by means of a panel of 10 markers that span approximately 60 cM in the 11q22-23 region centered around S144 and THY1. Location scores indicate that three contiguous subsegments within the [S144-THY1] segment, as well as three contiguous segments telomeric to THY1, are each unlikely to contain the AT locus, while the more centromeric [STMY-S144] segment is most likely to contain the AT locus. These data, together with recent refinements in the linkage and physical maps of 11q22-23, place the AT locus at 11q23.

Alleles↗
PubMed1989

Automated DNA sequencing methods involving polymerase chain reaction.

Polymerase chain reaction (PCR) as a method for preparing DNA templates has been used for several DNA sequencing applications. An in situ procedure for directly sequencing PCR products by the dideoxy-termination method has been developed by using fluorophore-labeled sequencing primers. Completed sequence reactions were combined and loaded into a single electrophoretic lane of a fluorescence-based DNA sequence analyzer. DNA targets devoid of a universal primer sequence could be sequenced with labeled universal primers by incorporating a universal primer sequence into the PCR product. With this method, the sequence of a 351-bp region in the bacteriophage lambda genome was fully analyzed in a single lane with automatic base identification accuracy of greater than 99%. An unknown sequence, 1.7 kb long, also was sequenced by this procedure, in combination with a "PCR gene walking" strategy. Comparison of the 1110 bases in overlapping sequence data from both strands yielded only two single-base ambiguities. Automated DNA sequence analysis of the highly polymorphic HLA-DQA-1 (alpha) region in the human genome can be performed with this simple methodology. Use of this PCR-sequencing method to analyze DNA extracted from a one-month-old blood sample from an individual who is heterozygous at this locus allowed unambiguous assignment of genotype.

Autoanalysis↗
PubMed1989

Use of deoxyribonucleic acid probes in the identification of cell origin and detection of cellular contamination in human lymphoblastoid cell lines.

Established lymphoblastoid cell lines have provided a valuable reference source for studying neoplastic lymphoproliferative disorders in humans. However, two major problems are associated with the establishment and growth of these cell lines: (a) the established cell line may not represent the original neoplastic clone, and (b) contamination of the established cell line with the other cell lines may occur. Lymphoblastoid cell lines "W" and "SP5" were established from splenectomy specimens of two patients with hairy cell leukemia. Both cell lines displayed B cell characteristics by immunophenotypic and Ig gene rearrangement studies. The banding patterns of the rearranged Ig genes (heavy and light chains) in the W cell line were different and in the SP5 cell line were identical with the corresponding untransformed splenic cell lines, indicating that cell line SP5 did and cell line W did not represent the original neoplastic clone. Continuous cultures of some of the subclones derived from cell line W and SP5 led to the growth of the cell lines W15T, W17T, and SP5T which all demonstrated T cell features based on immunophenotypic and T cell receptor rearrangement studies. However, the T cell receptor alpha and beta rearranged bands as well as bands generated by hybridization with highly polymorphic DNA probes p YNH24 and 0-3315-32 in these three lines and a human T cell leukemia line (CEM), were identical indicating that W15T, W17T and SP5T cell lines were contaminated with CEM. Studies of gene establishment patterns and DNA polymorphisms by Southern blotting are effective methods to establish clonal identity and to rule out cellular contamination in lymphoblastoid cell lines.

B-Lymphocytes↗
PubMed1988

DNA polymorphism in the human Thy-1 gene.

Thy-1 is a membrane glycoprotein expressed predominantly in brain tissue and occasionally in lymphoid tissue. The human Thy-1 gene is located on chromosome 11q22.3. Although two allelic forms of Thy-1 exist in mice (Thy-1.1 and Thy-1.2), no allelic forms have been described for the human Thy-1 gene. We describe a polymorphic MspI site within the human Thy-1 gene that distinguishes two alleles, 8 and 9, which are represented in a northern European population at frequencies of 0.7 and 0.3, respectively. Thy-1, therefore, provides a potentially useful marker to identify linkages with human disease genes located near 11q22.

Alleles↗
PubMed1987

Human ferritin genes: chromosomal assignments and polymorphisms.

The ferritins are a family of proteins that function intracellularly to sequester iron that otherwise would be toxic to the cell. The molecules are comprised of 24 heavy and light subunits, the heavy:light ratio varying widely in a tissue-specific manner. We cloned DNA sequences for both the heavy (HL217-1) and light (HL227) subunits from a cDNA library derived from messages that are strongly regulated during in vitro-induced differentiation of a promyelocytic leukemia cell line, HL-60, into either neutrophils or macrophages. The heavy-subunit family (FTH) includes 15-20 genes and/or pseudogenes; the light-subunit family (FTL) includes at least three genes. We have confirmed and extended the chromosomal localization of the heavy-subunit "genes" to chromosomes 1-6, 8, 9, 11, 13, 14, 17, and X. We have also identified and characterized two genetic polymorphisms: FTH/BamHI and FTH/TaqI. FTH/BamHI localizes to chromosome 3, is biallelic, and has a heterozygosity frequency of .39, a minor allele frequency of .33, and a polymorphic information content (PIC) of .34. FTH/TaqI is measured by the presence or absence of a single 6-kb fragment that is absent (i.e., "homozygosity" being presumed) in approximately 63% of Caucasians (PIC = .27). We discuss the possibility that gene-family probes that hybridize to many discrete members of dispersed gene families could be used in conjunction with pulsed- or inverted-field gels to screen a large number of specific genomic regions for microdeletions.

Alleles↗
PubMed1982

Overproduction of dihydrofolate reductase and gene amplification in methotrexate-resistant Chinese hamster ovary cells.

Stable isolates of Chinese hamster ovary cells that are highly resistant to methotrexate have been selected in a multistep selection process. Quantitative immunoprecipitations have indicated that these isolates synthesize dihydrofolate reductase at an elevated rate over its synthesis in sensitive cells. Restriction enzyme and Southern blot analyses with a murine reductase cDNA probe indicate that the highly resistant isolates contain amplifications of the dihydrofolate reductase gene number. Depending upon the parenteral line used to select these resistant cells, they overproduce either a wild-type enzyme or a structurally altered enzyme. Karyotype analysis shows that some of these isolates contain chromosomes with homogeneously staining regions whereas others do not contain such chromosomes.

Animals↗
PubMed1981

Insertion of new genes into bone marrow cells of mice.

Drug resistance genes such as those coding for a methotrexate-resistant dihydrofolate reductase (DHFR) or the thymidine kinase from herpes simplex virus can be used to confer a proliferative advantage on bone marrow cells of mice. As a result of this proliferative advantage, transformed cells become the predominant population in the bone marrow. Efficient gene expression was obtained for both the thymidine kinase and DHFR genes inserted into mouse bone marrow. Such gene insertion techniques may ultimately lead to the cure of life-threatening globinopathies such as sickle cell disease or the beta thalassemias. They may also be useful in reducing the hematopoietic toxicity of anticancer drugs.

Animals↗
PubMed1981

Complete nucleotide sequence of a chicken alpha-globin cDNA.

The entire sequence of a 541 bp insert in recombinant plasmid pHb1003 has been determined. This plasmid, which was shown to carry a cloned cDNA copy of the chicken alpha-globin mRNA, contains the complete structural gene as well as 19 bp of the 5'-untranslated region and 99 bp of the 3'-untranslated region. This sequence may encode a non-adult alpha-globin gene, especially since the cDNA clones were generated from phenylhydrazine-induced, globin-specific mRNA extracted from anemic white leghorns. The possibility that this alpha-globin might represent a stress globin is considered.

Amino Acid Sequence↗
PubMed1981

Human ribosomal RNA gene spacer sequences are found interspersed elsewhere in the genome.

A cloned EcoRI fragment containing human 18 S rRNA gene sequences was used to screen a gene library to obtain a set of 8 overlapping cloned DNA segments extending into the non-transcribed spacer region of the human ribosomal RNA gene cluster. 19.4 kb of the approx. 43-kb rDNA repeat was obtained in cloned form and mapped with restriction endonucleases. None of the clones obtained extended into 28 S rRNA sequences. A 7-kb region of non-transcribed spacer DNA shared in common between five independent clones was subjected to comparative restriction digests. It was estimated that sequences among the five different spacer isolated varied by not more than 1.0%, if all the observed differences are assumed due to point mutation. HaeII-restriction fragments from within this same 7-kb region contain sequences carried not only within the tandem repeats of the gene cluster but interspersed elsewhere in the genome. Some of these sequences correspond to the Alu family of highly repeated interspersed sequences.

Base Sequence↗
PubMed1980

Insertion of a new gene of viral origin into bone marrow cells of mice.

DNA containing the herpes simplex virus thymidine kinase (HSVtk) gene was used to transform wild-type tk+ mouse L cells to a tk++ status in vitro using methotrexate as a selective agent. HSVtk DNA was also used to transform mouse bone marrow cells in vitro. Transformed marrow cells injected into irradiated and methotrexate-treated recipient mice gave rise to proliferating cells which in some cases dominated the marrow population and which contained HSVtk gene sequences.

Animals↗
PubMed1980

Construction and characterization of a cDNA clone containing a portion of the bovine prolactin sequence.

Poly(A)-containing RNA from the bovine anterior pituitary has been used as a template for the enzymatic synthesis of double-stranded cDNA. The resulting double-stranded cDNA was inserted into the Pst I site of pBR322 with the oligo(dG)-oligo(dC) tailing technique and subsequently cloned in E. coli chi 1776. Clones containing sequences complementary to prolactin mRNA were identified by colony hybridization with partially purified prolactin cDNA. A 250 base pair sequence from one prolactin positive clone was extensively characterized and shown to contain the coding information for amino acids 119-192 of authentic bovine prolactin. The recombinant DNA from this clone was covalently attached to diazotized aminocellulose and used to purify prolactin mRNA from a mixture of mRNAs.

Animals↗
PubMed1980

Gene transfer in intact animals.

Resistance to methotrexate was induced in bone marrow cells of mice by transformation in vitro with DNA from a drug-resistant cell line. Transformed cells were injected in vivo and haematopoietic cells expressing resistance were selected by drug treatment of recipients. Transformed cells had elevated levels of dihydrofolate reductase and demonstrated a proliferative advantage over untransformed cells, indicating successful gene transfer.

Animals↗
PubMed1979

Sequence analysis of cloned cDNA encoding part of an immunoglobulin heavy chain.

The recombinant plasmid pH21-1 consists of mouse-derived complementary DNA (cDNA) in the E. coli plasmid pMB9. The mouse insertion has been completely sequenced, and encodes the CH3 domain and half the CH2 domain of the immunoglobulin gamma1 heavy chain. The predicted amino acid sequence differs at several positions from that previously published for this protein. The pattern of codon usage resembles that in some other eukaryotic messenger RNAs. A computer program has been used to predict the optimum secondary structure for the mRNA encoding the CH3 domain and the inter-domain junction.

Animals↗
PubMed1979

Isolation of human germ-line DNA suitable for recombinant DNA studies.

Dna from human sperm cells can be isolated with size and purity sufficient for use in recombinant DNA research. The DNA averages 100 000 base pairs (bp) in size (about 70.10(6) daltons) and is free of somatic cell, bacterial, and viral DNA. It can therefore be cloned under P2 + EK2 conditions as stipulated in the 1978 NIH Guidelines for recombinant DNA research.

Centrifugation, Density Gradient↗