Preparation of template for automated sequencing using QIAGEN resin.
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Autoanalysis↗
Biomedical subjects
S W Hunt
Publications and source records attributed to S W Hunt.
Noncollagenous (NC1) domain of collagen VII resembles multidomain adhesion proteins involved in tissue-specific organization of extracellular matrix.
Type VII collagen (C7) is a stratified squamous epithelial basement membrane protein composed of three identical alpha chains, each consisting of a 145-kDa amino-terminal noncollagenous (NC1) domain and a 145-kDa carboxyl-terminal collagenous domain. Morphologic and biochemical studies have shown that tissue-specific aggregates of C7 dimers called anchoring fibrils may contribute to epithelial basement membrane organization and adherence by interacting with extracellular matrix (ECM) proteins such as type IV collagen. In this study, we cloned a cDNA encoding most of the NC1 domain of C7. The deduced amino acid sequence revealed motifs characteristic of multidomain ECM proteins that contribute to the tissue-specific organization of ECM including a region of 7 1/2 sequential fibronectin type III (Fn III) homology repeats, a potential collagen-binding region homologous to the A domain of von Willebrand factor (vWf) and an RGD sequence. A purified C7 fusion protein containing these motifs specifically bound to type IV collagen in a functional assay. These results suggest that regions within the NC1 domain of C7 mediate interactions with lamina densa and dermal ECM proteins including type IV collagen. Structural mutations and autoepitopes in these regions may represent mechanisms for the development of defective basement membrane organization and adherence in genetic and autoimmune forms of epidermolysis bullosa.
Amino Acid Sequence↗
Analysis of MHC class I gene expression in adult bone marrow and fetal liver of the BALB/c mouse.
The BALB/c mouse has at least 29 class I genes encoded in the Qa, Tla, and Hmt (histocompatibility dependent on a maternally transmitted factor) regions of the MHC. The pattern of expression of these class Ib MHC genes is not well characterized, although some of their products such as the serologically detectable Qa-2 and TL Ag are expressed mainly in lymphoid and hematopoietic tissues. In this study, the expression pattern of BALB/c class I genes has been analyzed in adult bone marrow and fetal liver. cDNA libraries were synthesized from these tissues, and isolated class I cDNA clones were characterized by hybridization with oligonucleotide probes and by nucleotide sequence analysis. Of the 29 total class Ib genes, transcripts of five including Q6d, Q7d, T9c, T10c, and the 37 gene were isolated from the bone marrow cDNA library. Four of these can encode proteins; the sequence of the T10c gene demonstrates it is most likely a pseudogene. A non-overlapping set of three class Ib cDNA clones was obtained from the fetal liver, including T13c, the Thy 19.4 gene, and a previously uncharacterized class I gene provisionally designated as FL 57.2. Although the majority of H-2Dd cDNA clones that were analyzed lack introns, many of the class Ib cDNA clones contain intron sequences. This suggests that the expression of some of these genes may be regulated at the level of RNA splicing. The T13c gene encodes the thymus leukemia Ag in BALB/c mice. We have confirmed that the T13c gene is expressed in fetal liver by flow cytometric analysis of cells stained with anti-TL mAb.
Animals↗
Synthesis of complement component C5 by human B and T lymphoblastoid cell lines.
Human B and T lymphoblastoid cell lines were shown to synthesize C5. C5 synthesis was quantitated with an enzyme-linked immunosorbent assay (ELISA) that utilized a pool of C5-specific monoclonal antibodies (mAbs). Some level of C5 synthesis was detected in all eight of the B and T cell lines examined. In three of the cell lines, C5 was detected in both culture supernatants and whole cell detergent lysates, whereas in the other five cell lines, C5 was detected only in the cell lysates. Lymphoblastoid cells with both distributions of C5 were shown to synthesize a messenger RNA that was similar in size to the C5 mRNA expressed by the HepG2 hepatoma cell line. Estimates of the concentration of the C5 transcript in poly(A)+ RNA from lymphoblastoid and HepG2 cells suggested that C5 mRNA levels in the lymphoblastoid cell lines were comparable and about one-tenth of the levels in HepG2 cells. Lymphoblastoid C5, isolated by immunoaffinity chromatography from the supernatants of 35S-labeled cultures, had the same subunit composition as plasma-derived C5, but had an alpha subunit of slightly smaller relative mass.
B-Lymphocytes↗
Revised nomenclature of mouse H-2 genes.
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Animals↗
Genetic and molecular mapping of the Hmt region of mouse.
We have mapped a new region of the mouse major histocompatibility complex (MHC) that contains the nuclear gene, Hmt, for the maternally transmitted antigen, Mta. The Hmt region of chromosome 17 lies between a recombinational breakpoint distal to Tla and another proximal to Tpx-1, thus including Pgk-2. A novel MHC class I gene fragment, R4B2, was cloned and mapped to this region as was another new class I gene, Thy19.4. Both lie proximal to Pgk-2, within the distal inversion in t-haplotypes. The presence of several other MHC class I genes in the Hmt region is predicted from analysis of the recombinants that define the region.
Amino Acid Sequence↗
Comparison of exon 5 sequences from 35 class I genes of the BALB/c mouse.
DNA sequences of the fifth exon, which encodes the transmembrane domain, were determined for the BALB/c mouse class I MHC genes and used to study the relationships between them. Based on nucleotide sequence similarity, the exon 5 sequences can be divided into seven groups. Although most members within each group are at least 80% similar to each other, comparison between groups reveals that the groups share little similarity. However, in spite of the extensive variation of the fifth exon sequences, analysis of their predicted amino acid translations reveals that only four class I gene fifth exons have frameshifts or stop codons that terminate their translation and prevent them from encoding a domain that is both hydrophobic and long enough to span a lipid bilayer. Exactly 27 of the remaining fifth exons could encode a domain that is similar to those of the transplantation antigens in that it consists of a proline-rich connecting peptide, a transmembrane segment, and a cytoplasmic portion with membrane-anchoring basic residues. The conservation of this motif in the majority of the fifth exon translations in spite of extensive variation suggests that selective pressure exists for these exons to maintain their ability to encode a functional transmembrane domain, raising the possibility that many of the nonclassical class I genes encode functionally important products.
Amino Acid Sequence↗
Analysis of D2d: a D-region class I gene.
The mouse major histocompatibility complex is composed of several genes arranged into the K, D, Qa, and Tla regions. The D region of the BALB/c mouse includes genes D2d, D3d, and D4d, in addition to H-2Dd and H-2Ld. We have determined the DNA sequence of the D2d gene and compared it with the known sequences of several class I genes. The exon/intron structure of the D2d gene is similar to other class I genes. It also contains similar 5' regulatory elements. A frameshift occurs in exon seven, resulting in a gene product with a truncated cytoplasmic tail. To examine the surface expression of the D2d molecule, we generated an exon-shuffled construct containing the promoter and exons 1-3, encoding the signal peptide, alpha 1, and alpha 2 external domains of the D2d gene linked to exons 4-8, encoding the alpha 3, transmembrane and cytoplasmic domains, of the H-2Dd gene. The construct was transfected into mouse L cells, and a protein was detected at the cell surface by a monoclonal antibody (mAb) specific for the alpha 3 domain of H-2Dd, as well as by other class I-specific mAbs. Although D2d is expressed at low levels, it may be a functional class I gene that most probably evolved from a Qa region gene.
Amino Acid Sequence↗
Expression of the Q8/9d gene by T cells of the mouse.
The Q genes, specifying Qa antigens and situated in the extended part of the major histocompatibility complex (MHC) of the mouse, comprise a subgroup of MHC class I genes whose significance and function are still largely unknown. In screening a cDNA library made from the BALB/c inducer T-cell line Cl.Ly1-T1, we isolated 11 clones representing Q8/9, but none representing Q6 or Q7. Confirmatory evidence is given that the Q8/9 gene originated from fusion of the 5' region of the Q8 gene with the 3' region of the Q9 gene at a recombination site or hot spot in the vicinity of intron 4. Contrary to previous impressions that Q8/9 is an inert pseudogene, we find that the Q8/9 gene can be functional and encode a Qa-2, 3 antigen. One variety of the 11 Q8/9 clones isolated lacked exon 5, which encodes the transmembrane domain of class I glycoproteins, and thus may account for secretion of a soluble form of Qa-2, 3 antigen thought to be released by activated T cells.
Amino Acid Sequence↗
Analysis of a new class I gene mapping to the Hmt region of the mouse.
The major histocompatibility complex (MHC) of the BALB/c mouse contains three genes encoding classical class I molecules, as well as at least 32 nonclassical class I genes. Although much is known about the genes encoding the classical class I molecules, the majority of the nonclassical genes have not been characterized. This report describes a newly identified nonclassical class I gene, Thy19.4, which contains an open reading frame and resembles in several regards the genes encoding classical class I molecules. The similarities include shared amino acid sequence motifs which suggest that the putative Thy19.4 molecule may assume a tertiary structure similar to that of the classical class I molecules, as well as widespread transcription in a variety of tissues. However, unlike the classical class I genes, the Thy19.4 gene maps approximately 1 cm distal to the Tla region of the MHC, in the same region as the gene encoding the Hmt element of the maternally transmitted antigen.
Amino Acid Sequence↗
A low polymorphic mouse H-2 class I gene from the Tla complex is expressed in a broad variety of cell types.
We have previously described the isolation of pH-2d-37, a cDNA clone that encodes a so far unknown, poorly polymorphic, class I surface molecule. We report here the isolation of the corresponding gene, its nucleotide sequence, and its localization in the Tla region of the murine MHC. Using a RNase mapping assay, we have confirmed that the second domain coding region of the 37 gene displays very limited polymorphism, and that the gene is transcribed in a broad variety of cell types, in contrast to the genes encoding the known Qa and TL antigens. Possible functions are discussed.
Animals↗
Myelin deficient mice: expression of myelin basic protein and generation of mice with varying levels of myelin.
Mice homozygous for the mutation myelin deficient (mld), an allele of shiverer, exhibit decreased CNS myelination, tremors, and convulsions of progressively increasing severity leading to an early death. In this report we demonstrate in mld mice that the gene encoding myelin basic protein (MBP) is expressed at decreased levels and on an abnormal temporal schedule relative to the wild-type gene. Southern blot analyses, field-inversion gel electrophoresis studies, and analyses of mld MBP cosmid clones indicate that there are multiple linked copies of the MBP gene in mld mice. We have introduced an MBP transgene into mld mice and found that myelination increases and tremors and convulsions decrease. Mld and shiverer mice with zero, one, or two copies of the MBP transgene express distinct levels of MBP mRNA and myelin. The availability of a range of mice expressing graded levels of myelin should facilitate quantitative analysis of the roles of MBP in the myelination process and of myelin in nerve function.
Alleles↗
Structure of a gene encoding a murine thymus leukemia antigen, and organization of Tla genes in the BALB/c mouse.
We have determined the DNA sequence of a gene encoding a thymus leukemia (TL) antigen in the BALB/c mouse, and have more definitively mapped the cloned BALB/c Tla-region class I gene clusters. Analysis of the sequence shows that the Tla gene is less closely related to the H-2 genes than H-2 genes are to one another or to a Qa-2,3-region genes. The Tla gene, 17.3A, contains an apparent gene conversion. Comparison of the BALB/c Tla genes with those from C57BL shows that BALB/c has more Tla-region class I genes, and that one of the genes absent in C57BL is gene 17.3A.
Alleles↗
Deoxycoformycin resistant mammalian cells that overproduce adenosine deaminase.
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Adenosine Deaminase↗
Amplification of adenosine deaminase gene sequences in deoxycoformycin-resistant rat hepatoma cells.
Deoxycoformycin-resistant rat hepatoma cells exhibit up to a 2000-fold increase in adenosine deaminase activity compared to the sensitive parental cells. The increased enzyme activity in these cells is accompanied by similar increases in 1) the amount of adenosine deaminase protein, 2) the relative rate of adenosine deaminase synthesis in vivo, and 3) adenosine deaminase mRNA activity. To further investigate the mechanism(s) responsible for the overproduction of adenosine deaminase in these cells, we have isolated a recombinant plasmid containing a 1.4-kilobase insert complementary to at least part of the adenosine deaminase mRNA. Using this cDNA as a specific hybridization probe, all deoxycoformycin-resistant variants were shown to have increased amounts of adenosine deaminase mRNA and gene sequences. The relative increase in the level of mRNA and gene copy number was similar to the relative increase in enzyme activity for most resistant cell lines. However, the degree of adenosine deaminase gene amplification in one deoxycoformycin-resistant cell line (6-10-200) was 3-4-fold less than the relative increase in adenosine deaminase mRNA. These results indicate that the increased adenosine deaminase activity in deoxycoformycin-resistant rat hepatoma cells is due in large part, but not exclusively, to gene amplification.
Adenosine Deaminase↗
Increased adenosine deaminase synthesis and messenger RNA activity in deoxycoformycin-resistant cells.
The basis for the increased adenosine deaminase activity in deoxycoformycin-resistant rat hepatoma cells was investigated. Three variant cell lines with different levels of adenosine deaminase activity showed increases in the relative rate of synthesis of the enzyme in vivo. No difference in the rate of degradation of the enzyme was seen between the parental cell line and one variant cell line which exhibits a 180-fold increase in adenosine deaminase activity. Polysomal RNA isolated from this variant exhibited a 175-fold increase in the ability to direct the synthesis of adenosine deaminase in vitro.
Adenosine Deaminase↗
Evidence for a trans-dominant regulator of purine nucleoside phosphorylase expression in rat hepatoma cells.
Purine nucleoside phosphorylase (PNP) levels are modulated during the growth cycle of rat hepatoma cells and increase two- to three-fold as cells go from early exponential growth phase to stationary growth phase. A mutant of these hepatoma cells has been isolated which is deficient in PNP activity. Quantitative immunoprecipitation tests indicate that the decrease in enzyme activity is due to a decrease in the number of PNP molecules. The low level of PNP enzyme produced by the mutant, however, is indistinguishable from the wild-type enzyme, suggesting that the mutant may be defective in the ability to modulate PNP levels. Fusion of the mutant cells to wild-type parental cells results in hybrids that express the mutant phenotype. Segregants that arise from the hybrids show chromosome loss and reexpression of the wild-type parental phenotype, the mutant parental phenotype, and a 2S wild-type phenotype. These indicate the following about the defect in modulation in the mutant PNP-100: (1) it is trans dominant to the wild-type; (2) its effect is negative; (3) some genomic element is required for its continued effect; and (4) it does not act by obliterating its functioning counterpart in hybrid cells.
Animals↗
Adenosine deaminase from deoxycoformycin-sensitive and -resistant rat hepatoma cells. Purification and characterization.
Deoxycoformycin-resistant rat hepatoma cells exhibit up to 300-fold increase in adenosine deaminase activity compared to the sensitive parental cells. In order to determine the basis of the increased enzyme activity in deoxycoformycin-resistant cells, adenosine deaminase was purified from rat liver and deoxycoformycin-sensitive and -resistant cells. Physical, kinetic, and immunological properties of the purified enzymes were compared. Purified adenosine deaminase from all sources was found to be a monomer with an Mr approximately 45,000. In addition, the purified enzymes had a similar isozyme pattern in nondenaturing polyacrylamide gels. Km values for adenosine and Ki values for deoxycoformycin did not differ among the purified enzymes. By double diffusion analysis and quantitative immunoprecipitation, the purified enzymes were found to be immunologically indistinguishable. These data indicate that deoxycoformycin-resistant rat hepatoma cells produce increased amounts of adenosine deaminase protein which results in increased enzymatic activity.
Adenosine Deaminase↗