Identification of a cDNA encoding a long form of prolactin receptor in human hepatoma and breast cancer cells

Human PRL receptor cDNA clones from hepatoma (Hep G2) and breast cancer (T-47D) libraries were isolated by using a rat PRL receptor cDNA probe. The nucleotide sequence predicts a mature protein of 598 amino acids with a much longer cytoplasmic domain than the rat liver PRL receptor. Although this extended region has additional segments of localized sequence identity with the human GH receptor, there is no identity with any consensus sequences known to be involved in hormonal signal transduction. This cDNA will be a valuable tool to better understand the role of PRL in the development and growth of human breast cancer.     

Identification of multiple isoforms of the low-affinity human IgG Fc receptor

Two varieties of similar, but structurally distinct, cDNA clones for the human low-affinity receptors for the Fc portion of immunoglobulin G (FcγRII) have been isolated. One type of clone was obtained from human B lymphocytes, and the other from PHA-activated peripheral T cells and monocytes. Transfection of both prototype clones into Cos-7 cells and subsequent specific staining with monoclonal antibodies of the CDw32 group confirmed the identification of the gene products. The nucleotide sequence of the cDNA clone from B lymphocytes contains an open reading frame that encodes a protein of relative mass (M _r) 27000 with an extracellular domain of 179 amino acids containing three potential N-glycosylation sites, a 26 amino acid transmembrane domain, and a 44 amino acid cytoplasmic domain. The clones from peripheral T cells and monocytes both encoded a protein ofM _r 31000 with a 179 amino acid extracellular domain containing two potential N-glycosylation sites and a 26 amino acid transmembrane domain. The two types of clones had similar sequences in their immunoglobulin-like extracellular and transmembrane domains, but differed in their leader sequences and 3′-untranslated regions. The most notable difference between the clones was the presence of a distinctive 76 amino acid cytoplasmic domain in those isolated from T cells and monocytes.     

The dominant MHC class I gene is adjacent to the polymorphic<Emphasis Type="Italic"> TAP2</Emphasis> gene in the duck,<Emphasis Type="Italic"> Anas platyrhynchos</Emphasis>

We are investigating the expression and linkage of major histocompatibility complex (MHC) class I genes in the duck (Anas platyrhynchos) with a view toward understanding the susceptibility of ducks to two medically important viruses: influenza A and hepatitis B. In mammals, there are multiple MHC class I loci, and alleles at a locus are polymorphic and co-dominantly expressed. In contrast, in lower vertebrates the expression of one locus predominates. Southern-blot analysis and amplification of genomic sequences suggested that ducks have at least four loci encoding MHC class I. To identify expressed MHC genes, we constructed an unamplified cDNA library from the spleen of a single duck and screened for MHC class I. We sequenced 44 positive clones and identified four MHC class I sequences, each sharing approximately 85% nucleotide identity. Allele-specific oligonucleotide hybridization to a Northern blot indicated that only two of these sequences were abundantly expressed. In chickens, the dominantly expressed MHC class I gene lies adjacent to the transporter of antigen processing (TAP2) gene. To investigate whether this organization is also found in ducks, we cloned the gene encoding TAP2 from the cDNA library. PCR amplification from genomic DNA allowed us to determine that the dominantly expressed MHC class I gene was adjacent to TAP2. Furthermore, we amplified two alleles of the TAP2 gene from this duck that have significant and clustered amino acid differences that may influence the peptides transported. This organization has implications for the ability of ducks to eliminate viral pathogens.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers AY294416–22     

Isolation of cDNAs encoding the CD19 antigen of human and mouse B lymphocytes. A new member of the immunoglobulin superfamily

The CD19 (B4) molecule is a m.w. 95,000 cell-surface protein of human B lymphocytes that is expressed before Ig and persists throughout differentiation. In this report, cDNA clones that encode the CD19 molecule were isolated and the amino acid sequence of CD19 was determined. A cDNA clone that selectively hybridized to RNA from CD19+ cell lines was selected from a human tonsilar cDNA library using differential hybridization. This cDNA was used to isolate additional cDNA clones. Four of the five longest cDNA clones isolated were sequenced and found to contain unique sequences presumed to be introns. One clone, pB4-19, was near full length (2.1 kb) and did not contain these putative introns. pB4-19 contained an 1685 bp open reading frame that could encode a protein of about 60 kDa. COS cells that were transfected with pB4-19 expressed a nascent cell surface structure reactive with the anti-B4 antibody. Immunoprecipitation of this structure from surface-iodinated COS cells with the anti-B4 antibody revealed a m.w. 85,000 protein. Northern blot analysis indicated that pB4-19 hybridized with a predominant mRNA species of 2.4 kb and a minor species of 1.5 kb, found in only CD19+ cells. The pre-B cell line, PB-697, also expressed four larger RNA species that hybridized with pB4-19. cDNA clones that encode the putative cytoplasmic portion (247 amino acids) of the mouse CD19 molecule were also isolated and found to be highly homologous (79 and 75%) with the human CD19 nucleotide and amino acid sequences. The deduced amino acid sequence of the CD19 cytoplasmic tail shared no significant homology with other known proteins but the putative extracellular region contained two Ig-like domains indicating that CD19 is a new member of the Ig superfamily.     

Isolation and Sequence of Partial cDNA Clones of Human L1: Homology of Human and Rodent L1 in the Cytoplasmic Region

We have isolated cDNA clones coding for the human homologue of the neuronal cell adhesion molecule L1. The nucleotide sequence of the cDNA clones and the deduced primary amino acid sequence of the carboxy terminal portion of the human L1 are homologous to the corresponding sequences of mouse L1 and rat NILE glycoprotein, with an especially high sequences identity in the cytoplasmic regions of the proteins. There is also protein sequence homology with the cytoplasmic region of the Drosophila cell adhesion molecule, neuroglian. The conservation of the cytoplasmic domain argues for an important functional role for this portion of the molecule.     

Isolation and characterization of mouse CD7 cDNA

The human CD7 antigen is a glycoprotein, M _r 40 000, expressed on the surface of peripheral blood T-lymphocytes and thymocytes, and is the earliest surface antigen to appear on T-cell lineage cells. In this study, putative mouse CD7 cDNA was identified based on its similarities with human CD7. Five independent clones originating from the same mRNA species were isolated (designated as mCD7) by screening a mouse thymocyte cDNA library with human CD7 cDNA, J61, under moderate stringency. The longest insert of a 995 base pair had an open reading frame of 210 amino acids. Northern blot analysis using the mouse CD7 cDNA probe demonstrated a single 1.2 kilobase mRNA ni the thymus, spleen, bone marrow, and small intestine. The protein deduced from mCD7 cDNA consisted of the leader, extracellular, transmembrane, and cytoplasmic domains of 24, 126, 21, and 39 amino acids, respectively, based on the hydrophobicity plot and the structure of human CD7. The extracellualr domain contained three potential N-glycosilation sites, while the cytoplasmic domain contained one potential protein kinase C phosphorylation site. The amino acid sequence had 45.5% similarity with human CD7, while the similarities for the individual domains ranged from 49.2% to 63.2%. The six highly conserved regions, which may possibly be involved with still unknown CD7-mediated functions, were located in the extracellular and cytoplasmic domains.The nucleotide sequence data reported in this paper have seen submitted to the GenBank, DDBJ, and EMBL nucleotide sequence database and have been assigned the accession number D10329.     

The cDNA structure, expression, and chromosomal assignment of the mouse Fas antigen.

The cell surface Fas antigen is a membrane-associated polypeptide which can mediate apoptosis. cDNA clones encoding the Fas antigen were isolated from a cDNA library constructed with mRNA from the mouse macrophage cell line BAM3. The nucleotide sequence and the deduced amino acid sequence of the mouse Fas antigen were 58.5 and 49.3% identical, respectively, to the corresponding sequences of human Fas antigen cDNA. The mouse Fas antigen consists of 306 amino acids with a calculated Mr of 34,971 and contains a single transmembrane domain which divides the molecule into extracellular and cytoplasmic domains. A 2.1-kb mRNA coding for the Fas antigen was detected in the mouse thymus, heart, liver, and ovary but not in brain and spleen. The expression of the Fas antigen gene in mouse fibroblast L929 and macrophage BAM3 cell lines was significantly induced by treatment with IFN-gamma but not by IFN-alpha/beta. Interspecific backcross analysis indicated that the gene coding for the Fas antigen is in the distal region of mouse chromosome 19.     

Isolation and expression of cDNA encoding the murine homologues of CD1.

The cDNA encoding the murine CD1.1 and CD1.2 gene products were isolated and their complete nucleotide sequence was determined. The nucleotide sequence and genomic organization of these molecules were similar to human CD1. The sequences in the alpha 1- alpha 3 domains were almost identical to previously reported genomic clones from a different strain, indicating limited polymorphism among these molecules. The predicted amino acid sequence in the transmembrane region and in the cytoplasmic tail was identical for CD1.1 and CD1.2. The two cDNA were also homologous in the 5' untranslated region but diverged in the 3' untranslated region. In contrast to human CD1, which is expressed at high levels in thymus, the expression of CD1 message in murine thymus was not detected in either thymus leukemia Ag positive or negative strains. Cell expressing murine CD1.1 were generated after transfer of the CD1.1 cDNA into murine cell lines. Immunoprecipitation with a rat anti-mouse CD1.1 mAb showed that the transfected CD1 was expressed on the cell surface as a beta 2-microglobulin-linked heterodimer. These results demonstrate that the murine and human CD1 genes, although encoding homologous transmembrane glycoproteins, are expressed in distinct tissues and may serve different functions.     

Primary structures of two types of alpha-subunit of rat brain Na+,K+,-ATPase deduced from cDNA sequences

A rat brain cDNA library was screened by using as a probe a fragment of cDNA encoding the alpha-subunit of human Na+,K+-ATPase. Two different cDNA clones were obtained and analyzed. One of them was concluded to be a cDNA encoding the alpha-subunit of the weakly ouabain-sensitive rat kidney-type Na+,K+-ATPase. The deduced amino acid sequence consists of 1,018 amino acids. The alpha-subunit of the rat kidney-type Na+,K+-ATPase shows 97% homology in amino acid sequence with the alpha-subunit of human, sheep, or pig enzyme and 87% with that of Torpedo. Based on a comparison of the amino acid sequence at the extracellular domain of the alpha-subunit between weakly ouabain-sensitive rat kidney-type enzyme and the ouabain-sensitive human, sheep, pig, or Torpedo enzyme, it was proposed that only two significant amino acid replacements are unique to the rat kidney-type alpha-subunit. Another cDNA clone obtained showed 72% homology in nucleotide sequence with the former cDNA coding the alpha-subunit of the rat kidney-type Na+,K+-ATPase and the deduced amino acid sequence exhibited 85% homology with that of the alpha-subunit of rat kidney-type Na+,K+-ATPase.     

Evolution of major histocompatibility complex class I genes in Cetartiodactyls

Previous studies of cattle MHC have suggested the presence of at least four classical class I loci. Analysis of haplotypes showed that any combination of one, two or three genes may be expressed, although no gene is expressed consistently. The aim of this study was to examine the evolutionary relationships among these genes and to study their phylogenetic history in Cetartiodactyl species, including cattle and their close relatives. A secondary aim was to determine whether recombination had occurred between any of the genes. MHC class I data sets were generated from published sequences or by polymerase chain reaction from cDNA. Phylogenetic analysis revealed that MHC class I sequences from Cetartiodactyl species closely related to cattle were distributed among the main cattle gene "groups", while those from more distantly related species were either scattered (sheep, deer) or clustered in a species-specific manner (sitatunga, giraffe). A comparison between gene and species trees showed a poor match, indicating that divergence of the MHC sequences had occurred independently from that of the hosts from which they were obtained. We also found two clear instances of interlocus recombination among the cattle MHC sequences. Finally, positive natural selection was documented at positions throughout the alpha 1 and 2 domains, primarily on those amino acids directly involved in peptide binding, although two positions in the alpha 3 domain, a region generally conserved in other species, were also shown to be undergoing adaptive evolution.     

The complete sequence and expression patterns of the atrial myosin heavy chain in the developing chick

We have earlier reported partial cloning of a cDNA of a chick atrial myosin heavy chain (MHC) gene, CCSV2 and its expression pattern in embryonic chick hearts (Oana et al (1995) Eur J Cell Biol 67, 42-49). In this study, five overlapping cDNA clones (including CCSV2) which together encode the entire open reading frame of the chick atrial MHC gene were characterized, and both the entire nucleotide sequence consisting of 5825 bases and the deduced amino acid sequence consisting of 1931 amino acids determined. Reinvestigation of the nucleotide sequence of the previously reported and presumably different chick atrial specific MHC cDNA clone, AMHC1 (Yutzey et al (1994) Development 120, 871-883), revealed that our clone and AMHC1 encoded the same MHC. The chick atrial MHC gene was strongly expressed in developing chick atria from a very early stage (Hamburger and Hamilton stage 9, 29-33 h) to the adult stage. This gene was also expressed, although weakly, in the ventricle, somite (the precursor to skeletal muscle) and skeletal muscle during embryonic stages but not in adults.     

Molecular characterization,balancing selection,and genomic organization of the tree shrew (Tupaia belangeri) MHC class I gene

The major histocompatibility complex (MHC) class I genes play a pivotal role in the adaptive immune response among vertebrates. Accordingly, in numerous mammals the genomic structure and molecular characterization of MHC class I genes have been thoroughly investigated. To date, however, little is known about these genes in tree shrews, despite the increasingly popularity of its usage as an animal model. To address this deficiency, we analyzed the structure and characteristic of the tree shrew MHC class I genes (Tube-MHC I) and performed a comparative gene analysis of the tree shrew and other mammal species. We found that the full-length cDNA sequence of the tree shrew MHC class I is 1074 bp in length. The deduced peptide is composed of 357 amino acids containing a leader peptide, an α1 and α2 domain, an α3 domain, a transmembrane domain and a cytoplasmic domain. Among these peptides, the cysteines, CD8⁺ interaction and N-glycosylation sites are all well conserved. Furthermore, the genomic sequence of the tree shrew MHC class I gene was identified to be 3180 bp in length, containing 8 exons and 7 introns. In 21 MHC class I sequences, we conducted an extensive study of nucleotide substitutions. The results indicated that in the peptide binding region (PBR) the rate of non-synonymous substitutions (dN) to synonymous substitutions (dS) was greater than 1, suggesting balancing selection at the PBR. These findings provide valuable contributions in furthering our understanding of the structure, molecular polymorphism, and function of the MHC class I genes in tree shrews, further improving their utility as an animal model in biomedical research.     

Cloning and sequence analysis of beta-4 cDNA: an integrin subunit that contains a unique 118 kd cytoplasmic domain

The alpha 6 beta 4 complex is a member of the integrin superfamily of adhesion receptors. A human keratinocyte lambda gt11 cDNA library was screened using a monoclonal antibody directed against the beta 4 subunit. Two cDNAs were selected and subsequently used to isolate a complete set of overlapping cDNA clones. The beta 4 subunit consists of 1778 amino acids with a 683 amino acid extracellular domain, a 23 amino acid transmembrane domain and an exceptionally long cytoplasmic domain of 1072 residues. The deduced amino-terminal sequence is in good agreement with the published amino-terminal sequence of purified beta 4. The extracellular domain contains five potential N-linked glycosylation sites and four cysteine-rich homologous repeat sequences. The extracellular part of the beta 4 subunit sequence shows 35% identify with other integrin beta subunits, but is the most different among this class of molecules. The transmembrane region is poorly conserved, whereas the cytoplasmic domain shows no substantial identity in any region to the cytoplasmic tails of the known beta sequences or to other protein sequences. The exceptionally long cytoplasmic domain suggests distinct interactions of the beta 4 subunit with cytoplasmic proteins.     

Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry

Complementary DNA clones coding for the human secreted carbonic anhydrase isozyme (CA VI) have been isolated and their nucleotide sequences determined. These clones identify a 1.45-kb mRNA that is present in high levels in parotid submandibular salivary glands but absent in other tissues such as the sublingual gland, kidney, liver, and prostate gland. Hybridization histochemistry of human salivary glands shows mRNA for CA VI located in the acinar cells of these glands. The cDNA clones encode a protein of 308 amino acids that includes a 17 amino acid leader sequence typical of secreted proteins. The mature protein has 291 amino acids compared to 259 or 260 for the cytoplasmic isozymes, with most of the extra amino acids present as a carboxyl terminal extension. In comparison, sheep CA VI has a 45 amino acid extension [Fernley, R. T., Wright, R. D., & Coghlan, J. P. (1988b) Biochemistry 27, 2815]. Overall the human CA VI protein has a sequence identity of 35% with human CA II, while residues involved in the active site of the enzymes have been conserved. The human sheep secreted carbonic anhydrases have a sequence identity of 72%. This includes the two cysteine residues that are known to be involved in an intramolecular disulfide bond in the sheep CA VI. The enzyme is known to be glycosylated and three potential N-glycosylation sites (Asn-X-Thr/Ser) have been identified. Two of these are known to be glycosylated in sheep CA VI. Southern analysis of human DNA indicates that there is only one gene coding for CA VI.