Spleen cDNA Library Research Articles

Molecular Cloning of a Novel Cytoplasmic Protein Tyrosine Phosphatase PTPϵ

We have isolated a cDNA of a novel cytoplasmic variant of the protein tyrosine phosphatase ϵ (PTPϵ) from rat spleen cDNA library. Its deduced amino acid sequence of 642 residues was 94.6% identical to C-terminal 642 residues of human PTPϵ (HPTPϵ). However, N-terminal 12 amino acid residues of the rat PTPϵ had no homology to HPTPϵ. The unique N-terminal sequence of the rat PTPϵ was shorter and much more hydrophilic than the transmembrane domain of HPTPϵ. These results strongly suggest that the rat PTPϵ, designated here as PTPϵC, is a novel cytoplasmic PTP with two tandem catalytic domains. We also isolated by RT-PCR a rat transmembrane PTPϵ cDNA, designated as PTPϵM, which has the extracellular and transmembrane domains in addition to the common sequence to the PTPϵC. These results suggested that the PTPϵC and PTPϵM are generated from a single gene and may be involved in multiple functions in signal transduction.

Cloning and expression of a murine cDNA homologous to the human RCK/P54, a lymphoma-linked chromosomal translocation junction gene on 11q23

The RCK/P54 gene was previously isolated from the chromosome translocation breakpoint region on 11q23 of a human lymphoma cell line, RC-K8, with t(11;14)(q23;q32). It was found to encode a 472–483-amino-acid (aa) polypeptide belonging to an RNA helicase/translation initiation factor family. The aim of the present investigation was the isolation and comparative sequence analysis of the mouse RCK/P54 cDNA from a BALB/c spleen cDNA library. Sequence analysis revealed an open reading frame (ORF) predicting a polypeptide of 483 aa showing 97.7% identity to human RCK/p54. Northern analysis demonstrated a 7.5-kb signal in all tissues tested and in vitro translation analysis showed a 54-kDa polypeptide. These results indicate that both mouse and human RCK/P54 are highly conserved.

Primary Structure of a γ Subunit of G Protein, γ12, and Its Phosphorylation by Protein Kinase C

We have determined the primary structure of a novel gamma subunit (gamma 12, previously designated gamma S1) of G protein purified from bovine spleen. The mature gamma 12 protein composed of 68 amino acids had acetylated serine at the N terminus and geranylgeranylated/carboxylmethylated cysteine at the C terminus. This was consistent with the C-terminal prenylation signal in the amino acid sequence, which was predicted from gamma 12 cDNA isolated from a bovine spleen cDNA library. Western blots with the specific antibody against gamma 12 showed that gamma 12 is present in all tissues examined. Among various gamma subunits (gamma 1, gamma 2, gamma 3, gamma 7, and gamma 12), gamma 12 has a unique property to be phosphorylated by protein kinase C. The phosphorylated amino acid residue was Ser1 (or Ser2). The phosphorylated beta gamma 12 associated with Go alpha more tightly than the unphosphorylated form. Exposure of Swiss 3T3 and aortic smooth muscle cells to phorbol 12-myristate 13-acetate and NaF induced phosphorylation of gamma 12. Stimulation of aortic smooth muscle cells with natural vasoactive agents such as angiotensin II and vasopressin also induced phosphorylation of gamma 12. The extent of phosphorylation of beta gamma 12 in vitro was suppressed by a complex formation with Go alpha, which was relieved by the addition of guanosine 5'-O-(3-thiotriphosphate) or aluminum fluoride. These results strongly suggest that gamma 12 is phosphorylated by protein kinase C during activation of receptor(s) and G protein(s) in living cells.

Sequence of the rabbit glyceraldehyde-3-phosphate dehydrogenase-encoding cDNA

Two full-length cDNA clones encoding rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were isolated from a λgt10 rabbit spleen cDNA library and sequenced. As predicted from the open reading frame (ORF) in vitro translation of a sense orientation GAPDH cDNA clone yielded a protein product with a molecular mass of 37 kDa. Rabbit GAPDH exhibits a high degree of homology to the mouse, rat, hamster, chicken and human GAPDH on both the nucleotide (nt) and amino acid (aa) levels.

Identification and characterization of T-cell antigen receptor-related genes in phylogenetically diverse vertebrate species.

Characterization of the structure, multiplicity, organization, and cell lineage-specific expression of T-cell receptor (TCR) genes of nonmammalian vertebrate species is central to the understanding of the evolutionary origins of rearranging genes of the vertebrate immune system. We recently described a polymerase chain reaction (PCR) strategy that relies on short sequence similarities shared by nearly all vertebrate TCR and immunoglobulin (Ig) variable (V) regions and have used this approach to isolate a TCR beta (TCRB) homolog from a cartilaginous fish. Using these short PCR products as probes in spleen cDNA and genomic libraries, we were able to isolate a variety of unique TCR and TCR-like genes. Here we report the identification and characterization of a chicken TCR gamma (TCRG) homolog, apparent Xenopus and pufferfish TCR alpha (TCRA) homologs, and two horned shark TCR delta (TCRD)-like genes. In addition, we have identified what could be a novel representative of the Ig gene superfamily in the pufferfish. This method of using short, minimally degenerate PCR primers should speed progress in the phylogenetic investigations of the TCR and related genes and lend important insights into both the origins and functions of these unique gene systems.

Purification from placenta, amino acid sequence, structure comparisons and cDNA cloning of human glutaredoxin.

Glutaredoxin is generally a glutathione-dependent hydrogen donor for ribonucleotide reductase and also catalyses general glutathione (GSH)-disulfide-oxidoreduction reactions in the presence of NADPH and glutathione reductase. A Glutaredoxin from human placenta was purified to homogeneity, as judged by SDS/PAGE and IEF (12 kDa). Purification was monitored by the activity with hydroxyethyl disulfide as a substrate. Values of pI for glutaredoxin were obtained by IEF; the pI of the protein shifted from 7.3 in its fully reduced state to 9.0 in the oxidized state after treatment with excess hydroxyethyl disulfide. The glutaredoxin preparation showed GSH-dependent hydrogen-donor activity with recombinant mouse ribonucleotide reductase, it exhibited dehydroascorbate reductase activity as well as hydroxyethyl-disulfide-reducing activity. The amino acid sequence (residues 3-104) of glutaredoxin was determined by peptide sequencing and residues 1, 2 and 105 by cDNA sequence analysis. The glutaredoxin sequence comprised the classical active site for glutaredoxins -Cys22-Pro-Tyr-Cys25- and three additional half-cystine residues; two of these in positions 78 and 82. The sequence was similar to other known mammalian glutaredoxins (about 80% identities), with important differences such as one additional Cys residue (Cys7) and no Met residue. The sequence of human glutaredoxin was compared to that of Escherichia coli glutaredoxin with known three-dimensional structure in solution to identify conserved residues and predict a structure from alignment. In particular the GSH-binding site of glutaredoxin was conserved between all molecules. A cDNA that encodes the entire glutaredoxin gene (grx) and flanking sequences was isolated from a human spleen cDNA library. The nucleotide sequence of this cDNA (0.8 kb) was determined, including the complete grx gene.

T-cell receptor gene homologs are present in the most primitive jawed vertebrates.

The phylogenetic origins of T-cell immunity and T-cell antigen receptor (TCR) genes have not been established. A PCR approach using short, minimally degenerate oligodeoxynucleotide primers complementing conserved variable region segments amplifies TCR-like products from the genomic DNA of Heterodontus francisci (horned shark), a representative phylogenetically primitive cartilaginous fish. One of these products has been used as a probe to screen a Heterodontus spleen cDNA library and a clone was identified that is most related at the nucleotide sequence and predicted peptide levels to higher vertebrate TCR beta-chain genes. Genomic analyses of the TCR homologs indicate that recombining variable and joining region segments as well as constant region exons are encoded by extensive gene families, organized in the multicluster form, characteristic of both the immunoglobulin heavy- and light-chain gene loci in the cartilaginous fishes. Greater numbers of homologous products were identified when a probe complementing the putative constant region of the TCR homolog was used to screen the same cDNA library. A high degree of intergenic variation is associated with the putative variable region segments of these isolates. Direct evidence is presented for TCR-like genes, which presumably are associated with T-cell function, at the earliest stages in the phylogenetic emergence of jawed vertebrates.

Molecular cloning of rat JAK3, a novel member of the JAK family of protein tyrosine kinases

We have cloned and sequenced a cDNA (JAK3) encoding a novel member of the JAK family of protein tyrosine kinases. JAK3 was identified by RT-PCR of rat mesangial cells using degenerate oligonucleotide primers, and a full-length clone was isolated from a rat spleen cDNA library. The primary structure of JAK3 showed cDNA with an open reading frame of 1,100 amino acids which comprises the PTK catalytic domain and a second kinase-related domain characteristic for JAK kinase. JAK3 was phylogenetically shown to be most closely related to JAK2 among the previously known JAK family members, JAK1, JAK2 and Tyk2. Southern analysis revealed that JAK3 is a single copy gene and well conserved in the vertebral genome. Northern analysis indicated that the 4.0 kb mRNA was transcribed in a variety of tissues including spleen, lung, kidney and intestine.

Cloning, expression and tissue distribution of rat platelet‐activating‐factor‐receptor cDNA

The biological functions of platelet-activating factor (PAF) have been extensively studied in the rat. However, the precise structure and distribution of rat PAF receptor has not been reported. To address this question, we isolated a rat PAF-receptor cDNA from a size-fractionated rat spleen cDNA library. The deduced amino acid sequence of the rat PAF receptor showed 80% and 79% identity with guinea pig and human PAF receptors, respectively. Pharmacological properties (ED50, inhibition by WEB2086) of rat PAF receptors expressed in Xenopus oocytes were similar to those for PAF receptors expressed from guinea pig or human cDNAs. Northern blot analysis showed a widespread distribution of PAF-receptor mRNA in almost all organs including spleen, small intestine, kidney, lung, liver and brain. Considerable difference in the PAF-receptor distribution detected among species suggests the existence of a species-specific and tissue-specific regulatory mechanism for PAF-receptor-mRNA expression. Isolation of rat PAF-receptor cDNA should facilitate further analysis of PAF-receptor function and pharmacology in diverse pathophysiological processes.

Linkage of a new member of the lectin supergene family to chicken Mhc genes.

With the use of tissue-specific cDNA probes, several genes, which do not correspond to the class I (B-F), class II (B-L), or class IV (B-G) genes, were detected within the cosmid clusters containing the chicken major histocompatibility genes. We isolated cDNA clones with a probe corresponding to one of them, the 17.5 gene, located between two class I genes. The 17.5.3 cDNA, isolated from a chicken spleen cDNA library, encodes a 257-residue-long protein. This sequence shows significant similarity with several members of the C-type animal lectin superfamily and is probably a type II transmembrane protein. Analysis of several cDNA clones, together with Southern blot experiments, strongly suggest that this gene belongs to a multigene family, with at least some of its members being polymorphic. Several arguments lend support to the possibility that, together with the linked Mhc genes, the 17.5 gene is part of the recently described Rfp-Y system.

Complete cDNA sequence of a rainbow trout IgM gene and evolution of vertebrate IgM constant domains.

A complete cDNA clone encoding secreted IgM molecules was isolated from a spleen cDNA library of rainbow trout (Oncorhynchus mykiss). It encodes the leader peptide, VH, DH, JH, and the whole constant region of the secreted IgM molecule. We also studied the copy number of the IgM gene and an unusual RNA splicing mechanism which generates membrane IgM lacking the CH4 domain in rainbow trout. Amino acid sequence comparison of IgM protein sequences from this fish and other vertebrates indicates that some domains of IgM have evolved at a relatively constant rate. The evolution of salmonid fish including divergence time is discussed.

Molecular Cloning of the cDNA and Chromosomal Localization of the Gene for a Putative Seven-Transmembrane Segment (7-TMS) Receptor Isolated from Human Spleen

A family of proinflammatory cytokines sharing several structural features has been described and includes, for example, interleukin-8, monocyte chemoattractant protein-1, and melanocyte growth stimulatory activity. Recently, the receptors for interleukin-8 have been isolated and found to belong to the seven-transmembrane domain class of G protein-coupled receptors. As other members of this cytokine family likely interact with similar receptors, the polymerase chain reaction was employed to isolate related receptors from human peripheral blood adherent cells. Degenerate oligonucleotide primers based on the rabbit interleukin-8 receptor sequence were used. The corresponding full-length cDNA was isolated from a human spleen cDNA library. The predicted protein sequence of this clone, designated pBE1.3, was 93% identical to that of a cDNA isolated from bovine locus coeruleus, which apparently encodes a neuropeptide Y receptor, and also shows similarity with the interleukin-8 receptor and the human cytomegalovirus US28 sequences. The gene, designated D2S201E was localized to human chromosome 2q21. By Northern blotting, transcripts hybridizing to this cDNA were present in a variety of tissues and cells, including those of hemopoietic origin.

Binding of a protein tyrosine phosphatase to DNA through its carboxy-terminal noncatalytic domain

The noncatalytic domain of a non-receptor-type protein-tyrosine phosphatase (the T-cell phosphatase or PTP-S) isolated from a rat spleen cDNA library shows homology with the basic domains of transcription factors Fos and Jun [Swarup, G., Kamatkar, S., Radha, V., & Rema, V. (1991) FEBS Lett. 280,65-69]. We have expressed this phosphatase in Escherichia coli under the control of T7 promoter. The PTP-S gene product expressed in E. coli shows protein-tyrosine phosphatase activity and binds to DNA at pH 7.4 as determined by DNA affinity chromatography, Southwestern blotting, and gel retardation methods. The carboxy-terminal region of this phosphatase was fused with glutathione S-transferase by constructing expression vectors. Experiments using fusion proteins with glutathione S-transferase suggest that the carboxy-terminal 57 amino acids of PTP-S are sufficient for DNA binding. Deletion of the C-terminal 57 amino acids of PTP-S protein abolished its DNA binding property, as determined by Southwestern blotting, but not its enzymatic activity. This suggests that the C-terminal 57 amino acids are essential for the DNA binding function of this protein but not for its enzymatic activity. Another non-receptor-type protein-tyrosine phosphatase, PTP-1, when expressed in enzymatically active form in E. coli did not bind to DNA. These results suggest that a nontransmembrane protein-tyrosine phosphatase, PTP-S, binds to DNA in vitro through its carboxy-terminal noncatalytic region.

Cloning and nucleotide sequence of the cDNA encoding human erythrocyte-specific AMP deaminase

The nucleotide sequence of cDNA encoding human erythrocyte AMP deaminase has been determined by screening of human spleen cDNA library and by utilizing polymerase chain reaction (PCR) techniques. The 3.7 kb cDNA contains an open reading frame of 2301 bp which encodes 767 amino acids chain resulting in 89 kDa protein. The polyadenylation consensus signal (5′-AATAAA) located at 1212 bp 3′ downstream from the stop codon. The homologies to human and rat muscle-specific AMP deaminases showed 64.1% and 65.2% identities, respectively, at the nucleotide level in the area of open reading frame, and 60.2% and 59.8% similarities at the deduced amino acid level.

Expression of murine beta 7, alpha 4, and beta 1 integrin genes by rodent mast cells.

The screening of a rat mast cell cDNA library with a probe selected to recognize those genes preferentially or exclusively expressed by mast cells identified a rat gene sequence, RF-17, that shared homology with the beta-integrins. This integrin was expressed in rat tissues enriched for mast cells and T cells. The rat RF-17 sequence was used to isolate the murine homologue from a spleen cDNA library. The murine gene encodes a protein of 806 amino acids that is the probable homologue to the human beta 7 chain. Transcripts specific for the murine gene are found in the thymus, spleen, and lung. To attempt to identify the gene product for this new integrin chain, we examined the murine T cell line TK-1, which expresses a novel integrin heterodimer, lymphocyte Peyer's patch high endothelial venule adhesion molecule (LPAM-1), of a known alpha 4 chain and an unknown beta P chain, for expression of murine beta 7 (RF-17). This cell line expresses high levels of RF-17 transcripts, suggesting that beta P is encoded by the beta 7 gene. Bone marrow cells induced to differentiate into mast cells via IL-3 express the beta 7 gene as well as the genes encoding the murine integrin alpha 4 and beta 1 proteins. Surface staining analysis indicates that these cells express an alpha 4-containing integrin complex throughout the differentiation process. These data suggest that the Peyer's patch homing LPAM-1 receptor expressed by a subset of T cells consists of the beta 7 gene product and the alpha 4 chain, and that this integrin chain complex is also found on the surface of maturing mast cells. The presence of beta 1 transcripts also suggests that these maturing mast cells possess the LPAM-2 integrin complex (alpha 4/beta 1) as well. The experimental strategy described in this manuscript has, thus, identified a novel murine beta-integrin chain that is expressed by rodent T cells and mast cells.

Expression of interleukin-6 in porcine, ovine, and bovine preimplantation conceptuses.

A porcine interleukin-6 (pIL-6) cDNA has been cloned from pig spleen cDNA library to provide information that would allow us to study IL-6 mRNA expression during pregnancy of several domestic Artiodactyla. The cDNA is 1058 bp long and with a single open reading frame that encodes a 212 amino acid polypeptide with 28-residue signal sequence. It shares 61% and 43% amino acid sequence identity with human and mouse IL-6, respectively. PCR procedures with primers designed from regions of sequence conserved between human and pig have been used to identify IL-6 cDNA in lambda gt11 libraries constructed from day 15-16 (sheep), day 17 (cattle), and day 13-17 (pig) conceptus mRNA. The presence of IL-6 mRNA in elongating preimplantation ovine (days 13-25), porcine (days 13-21), and bovine (days 16-20) conceptuses was also demonstrated by PCR after reverse transcription of total ribonucleic acid with reverse transcriptase and by solution hybridization with a pIL-6 cRNA probe. These observations suggest that IL-6 is a product of these early conceptuses and may be involved in early maternal responses to the presence of an embryo within the uterus.

Characterization of a cDNA clone encoding for a porcine immunoglobulin μ chain

We present the complete sequence of cDNA clone BLM6, isolated from a porcine spleen cDNA library. BLM6 encodes for the constant region of a secreted μ heavy chain. The cDNA is 1342 bp in length, begins within Cμ1, and terminates in the 3′ untranslated region. The deduced amino acid sequence indicates there are 111 amino acids in Cμ2, 106 in Cμ3, and 113 in Cμ4, along with 19 carboxy-terminal residues that comprise the secreted terminus. The full length transcript is approximately 2.4 kb as determined by Northern blot analysis. Southern blot analysis suggests there is only one μ chain gene within the porcine genome. Comparison of the deduced amino acid sequence of porcine Cμ with other mammalian Cμ sequences indicates that the overall identity is approximately 60%, with the greatest identity seen in the secreted terminus (∼85%) and Cμ4 (∼70%); and less identity in Cμ3 (60%) and Cμ2 (50%).

Molecular cloning of putative members of the Na/H exchanger gene family. cDNA cloning, deduced amino acid sequence, and mRNA tissue expression of the rat Na/H exchanger NHE-1 and two structurally related proteins.

Biochemical and pharmacological data support the existence of multiple forms of the Na/H exchanger (NHE). Two isoforms, termed NHE-1 and NHE-2, have recently been isolated from rabbit ileal villus epithelial cells (Tse, C. M., Ma, A. I., Yang, V. W., Watson, A. J. M., Levine, S., Montrose, M. H., Potter, J., Sardet, C., Pouysségur, J., and Donowitz, M. (1991) EMBO J. 10, 1957-1967; Tse, C. M., Watson, A. J. M., Ma, A. I., Pouysségur, J., and Donowitz, M. (1991) Gastroenterology 100, A258). To identify additional molecular forms of the exchanger, rat brain, heart, kidney, stomach, and spleen cDNA libraries were screened for their presence using an NHE-1 cDNA probe under low stringency hybridization conditions. cDNAs encoding rat NHE-1 and two structurally related proteins, designated NHE-3 and NHE-4, have been isolated. Based on the deduced amino acid sequences, NHE-1, -3, and -4 are similar in size, having relative molecular masses of 91,506, 92,997, and 81,427, respectively. Overall, the proteins exhibit approximately 40% amino acid identity to each other and have similar hydropathy profiles, suggesting that they have the same transmembrane organization. The predicted N-terminal transmembrane regions of the three proteins, which span between 453 and 503 amino acids, exhibit the highest degree of identity (45-49%). In contrast, the C-terminal cytoplasmic regions, which span between 247 and 378 amino acids, exhibit very low amino acid identity (24-31%). Tissue distribution studies reveal that the NHE-1 mRNA is present at varying levels in all tissues examined, whereas NHE-3 and NHE-4 mRNAs exhibit a more limited distribution. NHE-3 mRNA is expressed at high levels in colon and small intestine, with significant levels also present in kidney and stomach. NHE-4 mRNA is most abundant in stomach, followed by intermediate levels in small intestine and colon and lesser amounts in kidney, brain, uterus, and skeletal muscle. These data suggest that the molecular basis for the functional diversity of the Na/H exchanger in mammals is based, at least in part, on expression of multiple members of a gene family.

A common ancestor of the mammalian transcription factors TCF‐1 and TCF‐1α/LEF‐1 expressed in chicken T cells

Several mammalian T cell-specific transcription factors have been cloned recently. Two of these, TCF-1 and TCF-1 alpha/LEF-1, display a moderate level of overall sequence similarity, and contain virtually identical versions of a novel type of DNA-binding domain, the HMG box. To study evolutionary aspects of the TCF transcription factors in relation to lymphoid differentiation, we have isolated chicken TCF clones from a spleen cDNA library. Low-stringency screening with human probes as well as a polymerase chain reaction-aided strategy resulted in the cloning of a single chicken TCF (chTCF) gene. Sequence comparison revealed that chTCF contained a TCF-1 alpha-like N terminus, and a TCF-1-like C terminus. Furthermore, TCF-1 and TCF-1 alpha were more homologous to chTCF than to each other. We postulate that chTCF is the direct descendant of a single ancestral gene, which has been duplicated in mammals to yield TCF-1 and TCF-1 alpha.

Secretion of mammalian ribonucleases from Escherichia coli using the signal sequence of murine spleen ribonuclease.

A nucleotide sequence identical with that of the recently identified murine pancreatic ribonuclease (RNAase) was isolated from a murine spleen cDNA library. Active RNAase was expressed and secreted from Escherichia coli lon-htpr- transformed with a plasmid containing the E. coli trp promoter followed by the murine RNAase gene sequence, including the original eukaryotic 26-amino-acid signal sequence. Approx. 1 mg of properly matured RNAase protein/litre was secreted into the medium of a fermentor culture after the promotor was induced by tryptophan starvation. When the signal sequence was deleted from the plasmid, intracellular RNAase activity was very low and there was no significant supernatant RNAase activity. Even higher RNAase yields were obtained with a synthetic gene for bovine pancreatic ribonuclease cloned after the signal sequence of the murine gene. About 2 mg of correctly processed RNAase A/litre was isolated from the growth medium, and a further 8-10 mg of correctly processed RNAase/litre could be isolated from the soluble fraction of the cells. Thus this eukaryotic signal sequence is both recognized by the E. coli transport and processing apparatus and gives efficient secretion, as well as export, of active, mature mammalian RNAases.