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Biomedical subjects

D Larhammar

Publications and source records attributed to D Larhammar.

At least 109 records · Page 6Linked to original sources
PubMed1987

Class II genes of the human major histocompatibility complex. Evolution of the DP region as deduced from nucleotide sequences of the four genes.

The DP region of the human major histocompatibility complex contains two alpha genes and two beta genes. The DP alpha 1 and beta 1 genes encode the expressed DP histocompatibility antigen molecule, while the DP alpha 2 and beta 2 genes are inactive in the haplotypes examined. Here we present the sequence of the two DP beta genes and of the expressed DP alpha 1 gene. Nucleotide sequence comparisons reveal a considerably greater degree of similarity between the two beta genes than between the two alpha genes. We propose that a duplication giving rise to the DP alpha gene pair evolutionarily preceded the corresponding DP beta gene duplication. We also propose, based on the orientation of other class II gene pairs, that the original DP molecule was encoded by the DP beta 1 and DP alpha 2 genes. At some stage during the evolution of the DP region both of the two pseudogenes appear to have been expressed.

Amino Acid Sequence↗
PubMed1987

Detection of neuropeptide Y and its mRNA in megakaryocytes: enhanced levels in certain autoimmune mice.

Neuropeptide tyrosine (neuropeptide Y, NPY) is a potent vasoconstrictor with a wide distribution in the central and peripheral nervous systems. Here we show that high levels of rat NPY mRNA are also found in peripheral blood cells, bone marrow, lung, and spleen. Furthermore, radioimmunoassay revealed high levels of NPY-like peptide in these tissues. In mice, the levels of splenic NPY mRNA and immunoreactive peptide differed extensively between strains and were greatly elevated in several strains (NZB, NZBxW, and BXSB) that develop a disease resembling human systemic lupus erythematosus. Like the rat, the NZB mouse showed a high content of NPY mRNA in peripheral blood cells and bone marrow. Immunohistochemical staining revealed NPY-like immunoreactivity in large cells morphologically identifiable as megakaryocytes in rat bone marrow and in the spleen of the NZB mouse strain. Expression of NPY mRNA in megakaryocytes in rat bone marrow and NZB mouse spleen was confirmed by in situ hybridization. These results indicate that NPY is synthesized in megakaryocytes, implying that NPY can be released from platelets and function as a vasoconstrictor during blood-vessel damage. In addition, the increase in splenic NPY in certain autoimmune mouse strains adds to the list of abnormalities associated with these strains.

Animals↗
PubMed1987

Structure and expression of the rat neuropeptide Y gene.

Neuropeptide Y is a 36-amino acid peptide that is abundant throughout the mammalian nervous system. It belongs to the same family of carboxyl-terminally amidated peptides as pancreatic polypeptide and peptide YY. We describe here the gene encoding the rat neuropeptide Y precursor. The gene spans 7.2 kilobase pairs and contains four exons. The exon organization is identical to the pancreatic polypeptide gene, although the amino acid sequences of the neuropeptide Y and pancreatic polypeptide precursors differ extensively. The predicted amino acid sequence of mature rat neuropeptide Y is identical to the human sequence. Also the sequence of the 30-amino acid carboxyl-terminal peptide of preproneuropeptide Y is highly conserved, which suggests that it is functionally important. Two neuropeptide Y alleles were found to differ at nine positions in 2.5 kilobase pairs at the 5' portion of the gene. No exon difference was found. One nucleotide substitution close to the gene promoter may influence the regulation of expression. Neuropeptide Y mRNA was found in all rat brain subregions tested, which shows that neuropeptide Y is synthesized throughout the brain. Developmentally, mRNA was detected in the rat brain as early as embryonic day 16 and increased rapidly to adult levels. The level of neuropeptide Y mRNA was also studied in several rat peripheral organs. Unexpectedly high levels were observed in heart and spleen. This mRNA may be synthesized in intrinsic ganglia and non-neuronal cells, respectively.

Alleles↗
PubMed1987

A molecular genetic approach to the identification of genes expressed predominantly in the neuroendocrine and immune systems.

Our results demonstrate that expression of neuropeptide tyrosine, one of the most abundant and widespread peptides in the mammalian nervous system, occurs in non-neuronal cells, in keeping with the emerging view that neuropeptide synthesis is not restricted to cells of the nervous system. RNA blot analyses and radioimmunoassays detected both NPY mRNA and NPY peptide in rat and mouse spleen, bone marrow, and peripheral blood cells. Immunohistochemical staining of sections from rat bone marrow with an NPY-specific antiserum revealed NPY-like immunoreactivity in megakaryocytes. In situ hybridization confirmed that the NPY-like peptide detected in megakaryocytes was synthesized de novo from NPY mRNA present in these cells. Megakaryocytes, the platelet-forming cells, originate from pluripotent hematopoietic stem cells present in the bone marrow as well as in the spleen of rodents. During microvascular damage, platelets aggregate at the damaged site and release bioactive substances. NPY is known to be a potent vasoconstrictor. Therefore, we propose that megakaryocyte-derived NPY is stored in platelets and released during platelet aggregation, resulting in a long-lasting vasoconstriction. Greatly elevated levels of megakaryocyte-derived NPY, as compared to the level found in BALB/C mice, were found in several mouse strains (NZB, NZB x W, and BXSB) which develop an autoimmune disease resembling systemic lupus erythematosus. Whether the elevation of megakaryocyte-derived NPY plays a role in the autoimmune disease progression in these mice or whether it merely reflects a related hematopoietic abnormality remains to be determined. Subtractive hybridization was used to isolate two cDNA clones that are predominantly expressed in the brain and the immune system. These and similarly derived cDNA clones will be used as molecular probes to study the mechanisms governing tissue-specific expression in the nervous and immune systems. Discovering the function of the proteins encoded by such cDNA clones may reveal evolutionary mechanisms shared by the nervous and immune systems, as well as a molecular basis for the interaction between these systems.

Animals↗
PubMed1986

Structure and expression of the chicken beta nerve growth factor gene.

The 3' exon of the chicken beta nerve growth factor (NGF) gene was isolated by the use of a murine cDNA probe. DNA sequence analysis of the clone suggests a mature chicken NGF protein of 118 amino acids, showing approximately 85% homology to mouse and human NGF. In addition to this conservation of the mature NGF, parts of the propeptide and the untranslated 3' end of the NGF gene are also highly homologous in chicken, human and mouse. Therefore, these sequences probably subserve important functions. Expression of NGF mRNA in various chicken tissues was examined by RNA blot analysis with a chicken NGF probe. A single mRNA of 1.3 kb was detected at high levels in heart and brain of 10-week-old roosters, and, at lower levels in spleen, liver and skeletal muscle. These data suggest a correlation between NGF expression and the density of sympathetic innervation in peripheral organs, in analogy with findings for mammalian tissues. In the adult avian brain, NGF mRNA is found at higher concentration in the optic tectum and cerebellum than in the cortex and hippocampus. This pattern of NGF expression differs from that previously described for the rat brain. During late stages of development (day 18), NGF mRNA was expressed both in heart and brain of embryos but at lower levels than in the adult.

Amino Acid Sequence↗
PubMed1986

Spontaneous insertions into cosmid vector: a warning.

During the course of characterization of a human genomic library we found that some of the selected pNNL cosmid clones carried only very short inserts. In addition, several clones that did contain full-length inserts were found to be unstable and generated repeated deletions of various portions of their inserts. Moreover, all the clones examined displayed rearrangements in the vector portions. The rearranged clones that were characterized by restriction mapping were found to have deletions starting between ClaI and HindIII in the region of the cos segment of the pNNL vector used in the construction of the library. We determined the nucleotide sequence of the 1300-bp EcoRI-PvuII segment located in the cos region, and by the computer search we found that it contains Escherichia coli insertion elements IS1.

Base Sequence↗
PubMed1986

Structure of the human Ia-associated invariant (gamma)-chain gene: identification of 5' sequences shared with major histocompatibility complex class II genes.

The human gene encoding the Ia-associated gamma (or invariant) chain was isolated by screening a genomic library in phage lambda with cDNA probes. The frequency of positive clones in the library, the overlapping restriction maps of the cloned fragments, and the patterns of genomic hybridization suggested that the gamma-chain gene exists as a single copy per haploid genome. The gene consists of 8 exons, spanning approximately 12 kilobases of DNA. All exon sequences were in an open reading frame, contained appropriate splice junction sequences, and encompassed the entire sequence of full-length gamma-chain mRNA, suggesting that the gene we isolated is most likely functional. Furthermore, "CAAT"-type and "TATA"-type promoter sequences were found at the expected positions upstream from the proposed cap site. The organization of the gamma-chain gene has none of the distinctive features of the immunoglobulin superfamily of genes, of which Ia alpha and beta chains are members. Therefore, the evolutionary origins, and perhaps the functions, of the Ia gamma chains are distinct from those of the other two Ia subunits alpha and beta. Despite the unrelatedness of these genes, consensus sequences found approximately 150 base pairs upstream from all the Ia alpha- and beta-chain genes sequenced to date were also found in analogous positions in the gamma-chain gene, suggesting a possible role in the coregulation of expression of these genes.

Base Sequence↗
PubMed1986

Expression of MHC class II antigens in human B-cell leukaemia, and increased levels of class II antigens and DR-specific mRNA after stimulation with 12-O-tetradecanoyl phorbol-13-acetate.

Cells from the peripheral blood of B-cell chronic lymphocytic leukaemia (CLL) patients were examined serologically for the expression of cell surface MHC class II antigens with monoclonal antibodies (mAbs) specific for the products of HLA-DP, -DQ and -DR genes, and mRNAs from the cells of three patients were analysed with a cDNA probe specific for DR beta chain genes. In 12 cases of CLL studied by indirect immunofluorescence and FACS analysis, a variable proportion of cells failed to express detectable levels of HLA-DP and HLA-DQ antigens at the cell surface, although greater than 90% of the cells had detectable expression of HLA-DR antigens. In all cases, greater than 90% of the cells expressed MHC class I antigens and the majority of cells reacted with the Leu-1 (CD5) mAb. Cells from different patients expressed variable levels of MHC class II antigens, and this was reflected in the finding of variable levels of mRNA detectable with the cDNA probe. Culture of cells with the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) induced much increased levels of expression of MHC class II antigens. HLA-DP and -DQ antigens were expressed on greater than 90% of the cells in all cases studied after culture of the cells with TPA, and MHC class II specific mRNA transcripts were correspondingly increased. In a single case of plasma cell leukaemia studied, MHC class II antigens were not detectable at the cell surface and their expression was not induced after culture of the cells with TPA.

Antibodies, Monoclonal↗
PubMed1985

Sequence of gene and cDNA encoding murine major histocompatibility complex class II gene A beta 2.

The murine major histocompatibility complex is known to express two class II molecules, A and E. They are composed of one alpha- and one beta-polypeptide chain. Recently, we reported the finding of an additional beta-chain second domain exon tentatively designated A beta 2. We describe here the nucleotide sequence of the A beta 2 gene and an A beta 2 cDNA clone. A beta 2 displays the same exon organization as the known expressed beta-chain genes, and the predicted A beta 2 polypeptide shows all the characteristic features of the expressed beta-chains. The predicted A beta 2 polypeptide shows 49-56% amino acid sequence identity to A beta and E beta chains and to the human DP, DQ, and DR beta-chains. These are 63-79% homologous to each other, indicating that A beta 2 is the most divergent member of the beta-chain family. The A beta 2 gene seems to be transcribed in the same types of cells as other class II genes. Detection of incompletely spliced A beta 2 mRNA and the finding of a cDNA clone containing an intron segment suggest that A beta 2 transcripts are processed slowly. Hybridizations with A beta 2 probes to restriction enzyme-digested genomic DNA indicate that the A beta 2 gene displays lower allelic polymorphism than the A beta gene.

Amino Acid Sequence↗
PubMed1985

Amino acid sequence homologies between rabbit, rat, and human serum retinol-binding proteins.

The main transporting protein for vitamin A in rabbit serum, the retinol-binding protein (RBP), was isolated and its amino acid sequence determined. Rabbit RBP was found to be highly homologous to human RBP, whose amino acid sequence was elucidated earlier, and to rat RBP. The rat RBP sequence was obtained by combining information deduced from the nucleotide sequences of two overlapping cDNA clones with the NH2-terminal sequence of the isolated protein determined by automated Edman degradation. The identity between the three proteins is approximately 90%. The high degree of homology between RBP molecules from different species is probably explained by the fact that RBP participates in at least three types of molecular interactions: in the binding of prealbumin, in the interaction with retinol, and in the recognition of a specific cell surface receptor. All these interactions should lead to a conservation of RBP structure. The amino acid differences between rabbit, rat, and human RBP are discussed in light of the recent elucidation of the three-dimensional structure of human RBP. Hybridization of a probe isolated from a rat RBP cDNA clone to restriction enzyme-digested genomic DNA from rat and mouse suggests that RBP is encoded by a single gene.

Amino Acid Sequence↗
PubMed1985

Characterization of an HLA DR beta pseudogene.

The class II molecules of the human major histocompatibility complex include the DR, DC, and SB antigens, each composed of an alpha and a beta polypeptide chain. We have isolated a DR beta gene in overlapping cosmid clones made from genomic DNA of a Dw4/DR4 homozygous individual. This gene consists of six exons and spans greater than 20 kilobases. Upon sequencing, it was found to possess several deleterious mutations, each capable of rendering the gene nonfunctional: (i) four splice junctions deviate from the G-T/A-G rule; (ii) two premature termination codons are present in the first domain exon; (iii) a 2-base-pair insertion causes a translational frame shift in the second domain exon. In addition, several amino acid residues that are conserved in all known expressed beta chains have been replaced in the amino acid sequence predicted from the pseudogene. Analysis of the pattern of nucleotide substitutions in the second domain exon suggests that most amino acid replacements occurred after the gene was inactivated. The inactivation may have been caused by insertion of a Kpn I repeat 5' to the promoter region, thereby interfering with transcription of the gene through removal of transcriptional enhancer elements. The DR beta pseudogene seems to be present also in other DR4 individuals.

Amino Acid Sequence↗
PubMed1984

Molecular map of the human HLA-SB (HLA-DP) region and sequence of an SB alpha (DP alpha) pseudogene.

The human major histocompatibility complex contains the genes for at least three different types of class II antigens, DR, DC and SB (DR, DQ and DP). They are all composed of an alpha and a beta chain. We have cloned a chromosomal region of 70 kb containing the SB (DP) gene family in overlapping cosmid clones. This segment contains two alpha genes and two beta genes, located in the order SB alpha 1, SB beta 1, SB alpha 2 and SB beta 2. The orientation of the alpha genes is reversed compared with that of the beta genes. This organisation suggests that the SB region has arisen by duplication of a chromosomal segment encompassing one alpha and one beta gene. Partial nucleotide sequences of the SB alpha 1 and SB beta 1 exons demonstrate that the genes correspond to SB alpha and beta cDNA clones. Consequently these genes are expressed. In contrast nucleotide sequence determination of the SB alpha 2 gene shows that it is a pseudogene.

Amino Acid Sequence↗
PubMed1984

Both alpha and beta chains of HLA-DC class II histocompatibility antigens display extensive polymorphism in their amino-terminal domains.

At least three class II antigens, all composed of an alpha and a beta subunit, are encoded in the human major histocompatibility complex, i.e., DR, DC and SB. Two cDNA clones, encoding a DC alpha and a DC beta chain, respectively, were isolated from a cDNA library of the lymphoblastoid cell line Raji (DR3,w6). The two polypeptides predicted from the nucleotide sequences of these clones are each composed of a signal peptide, two extracellular domains, a hydrophobic transmembrane region and a short cytoplasmic tail. Comparison of the DC alpha sequence with two previously published partial sequences shows that the majority of the differences is located in the amino-terminal domain. The differences are not randomly distributed; a cluster of replacements is present in the central portion of the amino-terminal domain. Likewise, the allelic polymorphism of the DC beta chains occurs preferentially in the amino-terminal domain, where three minor clusters of replacements can be discerned. The non-random distribution of the variability of DC alpha and beta chains may be due to phenotypic selection against replacement substitutions in the second domains of the polypeptides.

Amino Acid Sequence↗
PubMed1984

Mutations and selection in the generation of class II histocompatibility antigen polymorphism.

A comparison of seven human DR and DC class II histocompatibility antigen beta-chain amino acid sequences indicates that the allelic variation is of comparable magnitude within the DR and DC beta-chain genes. Silent and replacement nucleotide substitutions in six DR and DC beta-chain sequences, as well as in seven murine class II sequences (three I-A beta and four I-A alpha alleles) were analyzed. The results suggest that the mutation rates are of a comparable magnitude in the nucleotide sequences encoding the first and second external domains of the class II molecules. Nevertheless, the allelic amino acid replacements are predominantly located in the first domains. We conclude that a conservative selective pressure acts on the second domains, whereas in many positions in the first domains replacement substitutions are selectively neutral or maybe even favoured. Thus, the difference between the first and second domains as regards the number of amino acid replacements is mainly due to selection.

Amino Acid Sequence↗
PubMed1984

Signal sequences distinguish class II histocompatibility antigen beta chains of different loci.

The signal sequences of two HLA-DR beta chains and the DR alpha chain were determined. In addition, the major part of a DC beta-chain signal sequence was also elucidated. The data were obtained by combining amino acid sequence analyses of isolated alpha and beta chains with nucleotide sequencing of four cDNA clones. All signal sequences comprise 25 amino acids or more. The two HLA-DR beta-chain signal sequences are identical and exhibit only marginal homology to the DC beta-chain signal sequence. No homology is apparent between alpha- and beta-chain signal sequences. The differences in the signal sequences of the DR and DC beta chains suggest that these sequences may be used to assign beta chains to different loci of the human major histocompatibility complex.

Amino Acid Sequence↗