Isolation of a cDNA encoding a novel chicken chemokine homologous to mammalian macrophage inflammatory protein-1β

A cDNA encoding a novel chicken chemokine homologous to mammalian chemokine macrophage inflammatory protein 1β (MIP-1β) was isolated and characterised. The cDNA encodes a protein which is 75–80% homologous to human and mouse MIP-1β. All conserved amino acids characteristic of the mammalian chemokine family have been evolutionarily preserved in chicken MIP-1β, suggesting similar protein folding patterns and functional properties.     

Isolation, characterization, and chromosome mapping of a human A-C1 Ha-Ras suppressor gene (HRASLS)

Recently, we cloned a cDNA encoding a novel mouse protein, named A-C1, by differential display between two mouse cell lines, embryonic fibroblast C3H10T1/2 and chondrogenic ATDC5. Mouse A-C1 has homology with a ras-responsive gene, rat Ha-rev107 (Hrasls), and modulates a Ha-ras-mediated signaling pathway. Here, we report a cDNA encoding a human homolog of mouse A-C1. The deduced amino acid sequence of human A-C1 consists of 168 amino acids, and shows 83% identity with that of mouse A-C1. Human A-C1 mRNA was expressed in skeletal muscle, testis, heart, brain, and thyroid in vivo. Moreover, expression of human A-C1 mRNA was detected at a high level in human osteosarcoma-derived U2OS cells in vitro. By FISH analysis the human A-C1 gene (HRASLS) was mapped to human chromosome 3q28--> q29.     

Mouse and human GTPBP2, newly identified members of the GP-1 family of GTPase

We earlier identified the GTPBP1 gene which encodes a putative GTPase structurally related to peptidyl elongation factors. This finding was the result of a search for genes, the expression of which is induced by interferon-gamma in a macrophage cell line, THP-1. In the current study, we probed the expressed sequence tag database with the deduced amino acid sequence of GTPBP1 to search for partial cDNA clones homologous to GTPBP1. We used one of the partial cDNA clones to screen a mouse brain cDNA library and identified a novel gene, mouse GTPBP2, encoding a protein consisting of 582 amino acids and carrying GTP-binding motifs. The deduced amino acid sequence of mouse GTPBP2 revealed 44.2% similarity to mouse GTPBP1. We also cloned a human homologue of this gene from a cDNA library of the human T cell line, Jurkat. GTPBP2 protein was found highly conserved between human and mouse (over 99% identical), thereby suggesting a fundamental role of this molecule across species. On Northern blot analysis of various mouse tissues, GTPBP2 mRNA was detected in brain, thymus, kidney and skeletal muscle, but was scarce in liver. Level of expression of GTPBP2 mRNA was enhanced by interferon-gamma in THP-1 cells, HeLa cells, and thioglycollate-elicited mouse peritoneal macrophages. In addition, we determined the chromosomal localization of GTPBP1 and GTPBP2 genes in human and mouse. The GTPBP1 gene was mapped to mouse chromosome 15, region E3, and human chromosome 22q12-13.1, while the GTPBP2 gene is located in mouse chromosome 17, region C-D, and human chromosome 6p21-12.     

Cloning and expression of guinea pig TIMP-2. Expression in normal and hyperoxic lung injury

Tissue inhibitors of metalloproteinases (TIMPs) play a key regulatory role in extracellular matrix remodeling. By screening a lung library with a human TIMP-2 cDNA probe, we have isolated the cDNA corresponding to guinea pig TIMP-2. The 3.5-kb cDNA presents an open reading frame that predicts a protein of 220 amino acids showing 97.2, 96.8, 97.2, and 77.3% overall identity with human, mouse, rat, and chicken TIMP-2, respectively. Guinea pig TIMP-2 cDNA was expressed in CHO-K1 cells, showing a protein with the expected molecular weight and activity. Northern blot analysis revealed TIMP-2 expression in brain, kidney, intestine, spleen, heart, and lung. Transforming growth factor-beta downregulated TIMP-2 mRNA in guinea pig lung fibroblasts, whereas a variety of other stimuli showed no effect. In normal and hyperoxia-exposed lungs, TIMP-2 mRNA was mainly localized in alveolar macrophages and epithelial cells. No quantitative differences were found by Northern blot. These results confirm that TIMP-2 is highly conserved in mammals and largely expressed in lungs.     

Molecular cloning, expression and evolutionary analysis of the avian tyrosine kinase JAK1

The Janus protein tyrosine kinases (JAK) constitute a protein family that plays a pivotal role in signalling of a large number of cytokine receptors. The cDNA of the chicken homologue of JAK1 was cloned and its nucleotide sequence determined. Chicken JAK1 protein comprises 1150 amino acids as deduced from its cDNA sequence with a calculated molecular mass of 133 kDa. The overall structure of JAK proteins exemplified by the JAK homology domains JH1–JH7 is also preserved in chicken JAK1. Additionally, phylogenetic analysis demonstrates that chicken JAK1 is more closely related to mammalian JAK1 than to those of fish, exhibiting 80%, 79% and 63% identity in amino acid sequence to human, mouse and zebrafish JAK1, respectively. JAK1 proteins were found to be most conserved in the kinase (JH1) and pseudokinase (JH2) domains. This data is supported by Southern hybridization studies of ZOO blots. Chicken JAK1 shows a ubiquitous expression pattern and is transcribed as a 5.5 kb mRNA in various tissues and cell types. JAK1 expression was particularly high in lymphoid cells.     

Cloning and expression of the turtle (<Emphasis Type="Italic">Trachemys scripta</Emphasis>) immunoglobulin joining (J)-chain cDNA

The J-chain protein is a M(r) 15000 polypeptide associated with polymeric IgA and IgM. The complete cDNA sequences of human, mouse, cow, brushtail possum, chicken and frog J chains have been previously reported, but nothing is known about the cDNA and amino acid sequences of reptilian J chain. Here, we determined a turtle J-chain cDNA sequence by RT-PCR and RACE, and examined J-chain mRNA and protein expression by Northern blotting and immunohistochemistry. This turtle J-chain cDNA was 1934 bp and had an open reading frame of 477 nucleotides, encoding 159 amino acids. The mature J-chain protein is composed of 137 amino acids, M(r) approximately 15000. The deduced amino acid sequence of the turtle J chain was highly homologous to that of human (60%), mouse (61%), cow (60%), rabbit (60%), chicken (69%), brushtail possum (65%), Rana catesbeiana (47%) and Xenopus laevis (58%). Eight cysteine residues were located at the same positions as in these other species, with the exception of X. laevis. PROSITE database analysis indicated the presence of two N-glycosylation sites in turtle, one of which was novel. Northern blot analysis revealed that turtle J-chain mRNA was expressed in lung, stomach, spleen and intestine. In addition, immunohistochemistry showed J-chain-positive plasma cells in the intestine and spleen. These results suggest the presence of a mucosal immune system mainly composed of J-chain-containing Ig in the turtle.     

Molecular cloning, cDNA structure and tissue-specific expression of the human regulatory subunit RI beta of cAMP-dependent protein kinases.

Complementary DNA clones for the regulatory subunit RI beta of cAMP-dependent protein kinases were isolated from a human testis cDNA library using a mouse RI beta cDNA probe. One clone 2.4 kilobases (kb) in length contained an open reading frame of 1137 bases, and encoded a protein of 379 amino acids (excluding the initiator methionine). The human RI beta protein was one amino acid shorter than the corresponding protein in mouse and rat. The nucleotide similarity to mouse and rat sequences was 85.6% and 84.8%, respectively, while the amino acid similarity was 97.6% and 97.3%, respectively. Northern blot analyses revealed a 2.7 kb mRNA in human tissues and a 2.8 kb mRNA in mouse tissues. Both mouse and human RI beta mRNA were found to be expressed in most tissues, and not restricted to brain and testis as reported by others.     

Characterization of cDNA clones encoding a human fibroblast caldesmon isoform and analysis of caldesmon expression in normal and transformed cells

Overlapping cDNA clones encoding a low M gamma human nonmuscle caldesmon isoform (HUM 1-CaD) span the entire coding region (538 amino acids) as well as 111 base pairs (bp) of 5'-noncoding and 1249 bp of 3'-noncoding region. Northern blot probes derived from either the coding or 3'-noncoding region hybridized to a 4.3-kilobase mRNA in nonmuscle cells and a 5.2-kilobase mRNA in stomach tissue. Primer extension results indicated that the 5'-noncoding region of the HUM 1-CaD mRNA is approximately 700 bp in length and also suggested that 1-CaD mRNAs with common 5'-noncoding regions are expressed in both liver and fibroblast cells. Comparisons of the human, rat, and chicken 1-CaD amino acids sequences demonstrated that although each isoform has unique characteristics, extensive regions of conservation exist. Amino acids 27-53 and 97-127 are 100% identical in these isoforms while amino acids 297-531 of HUM 1-CaD are 94 and 85% identical to the rat and chicken 1-CaDs, respectively. In addition, the levels of HUM 1-CaD mRNA and protein appeared to be decreased by 2-4 fold in the transformed derivatives of KD and WI38 cell lines as judged by Northern and Western blot analysis. The results suggest that the decrease of 1-CaD protein in these transformed cells is a direct result of decreased 1-CaD mRNA synthesis and/or increased mRNA turnover.     

Human LAT1, a subunit of system L amino acid transporter: molecular cloning and transport function

We report here on the cloning and functional characterization of human LAT1, a subunit of the amino acid transport system L. The hLAT1 cDNA, obtained from a human placental cDNA library, codes for a protein of 507 amino acids. When functionally expressed in mammalian cells together with the heavy chain of the rat 4F2 antigen (r4F2hc), hLAT1 induces the transport of neutral amino acids. When expressed independently, neither hLAT1 nor r4F2hc was capable of amino acid transport to any significant extent. Thus, the hLAT1-r4F2hc heterodimeric complex is responsible for the observed amino acid transport. The transport process induced by the heterodimer is Na+ independent and is not influenced by pH. It recognizes exclusively neutral amino acids with high affinity. LAT1-specific mRNA is expressed in most human tissues with the notable exception of the intestine.     

Molecular cloning and DNA sequence analysis of cDNA encoding chicken homologue of the Bcl-2 oncoprotein.

We have isolated a 2228 bp cDNA clone encoding a chicken homologue of the human Bcl-2 oncoprotein by low-stringency hybridization screening of a lambda gt10 cDNA library derived from a chicken B-cell lymphoma. DNA sequence analysis of this cDNA revealed an open reading frame predicting a polypeptide of 232 amino acids and an M(r) of 25,839. The predicted protein is highly homologous to the human (73%) and mouse (70%) Bcl-2 proteins, and contains a hydrophobic stretch of amino acids within its carboxyl-end (213-229) consistent with an integral membrane protein. Areas of very high sequence homology shared by all three Bcl-2 proteins at the NH2-terminus (amino acids 1-33) and within the last 150 amino acids of these proteins suggest the presence of at least two evolutionarily conserved domains within the family of Bcl-2 proteins that may be important either for their targeting to mitochondria or their ability to block programmed cell death.     

Human microsomal xenobiotic epoxide hydrolase. Complementary DNA sequence, complementary DNA-directed expression in COS-1 cells, and chromosomal localization

A lambda gt11 expression library constructed from human liver mRNA was screened with an antibody against human microsomal xenobiotic epoxide hydrolase. The clone pheh32 contains an insert of 1742 base pairs with an open reading frame coding for a protein of 455 amino acids with a calculated Mr of 52,956. The nucleotide sequence is 77% similar to the previously reported rat xenobiotic epoxide hydrolase cDNA sequence. The deduced amino acid sequence of the human epoxide hydrolase is 80% similar to the previously reported rabbit and 84% similar to the deduced rat protein sequence. The NH2-terminal amino acids deduced from the human xenobiotic epoxide hydrolase cDNA are identical to the published 19 NH2-terminal amino acids of the purified human xenobiotic epoxide hydrolase protein. Northern blot analysis revealed a single mRNA band of 1.8 kilobases. Southern blot analysis indicated that there is only one gene copy/haploid genome. The human xenobiotic epoxide hydrolase gene was assigned to the long arm of human chromosome 1. Several restriction fragment length polymorphisms were observed with the human epoxide hydrolase cDNA. pheh32 was expressed as enzymatically active protein in cultured monkey kidney cells (COS-1).     

Molecular cloning and expression in gonad of Rana rugosa WT1 and Fgf9

Sry (sex-determining region on the Y chromosome) is required for testicular differentiation in mammals. In addition to Sry, other genes such as WT1, Fgf9, Dax1, Dmrt1 and Sox9 are widely accepted to be involved in the sex determination in vertebrates. However, the roles of these genes during sex determination still remain unclear in amphibians. This study was undertaken to examine the expression of WT1 and Fgf9 in the developing gonad of amphibians. We first isolated the WT1 cDNA from the frog Rana rugosa. Like WT1 in mice, R. rugosa WT1 showed 2 isoforms; i.e., one had an additional 3 amino acids, KTS, included between the third and fourth zinc fingers. However, 17 amino acids in exon 5 of mammalian WT1 could not be found in R. rugosa WT1, which is also the case in turtle and chicken. The mRNA of both isoforms (+KTS, -KTS) was detected in the lung, kidney and testis, but not in the ovary and muscle of adult frogs. The 2 isoforms were expressed first in the embryos at stage 23. Thereafter, the expressions remained constant in the gonad attached to mesonephros of both sexes during sex determination. We next isolated the R. rugosa Fgf9 cDNA encoding 208 amino acids. The amino acid sequence of Fgf9 had similarity greater than 92% with chicken, mouse and human Fgf9s, suggesting that Fgf9 is highly conserved among vertebrate classes. Fgf9 was expressed in the ovary of an adult frog strongly, but in the lung weakly. In contrast, the Fgf9 mRNA was hardly detected in the kidney, testis and muscle. Moreover, Fgf9 did not show a sexually dimorphic expression pattern during sex determination in R. rugosa. The results, taken together, suggest that both WT1 and Fgf9 are expressed in the indifferent gonad prior to sex determination without any difference in the expression between males and females. Thus, it seems unlikely that they are a key factor to initiate the divergence leading to testicular or ovarian differentiation in R. rugosa.     

Identification of a novel FGF, FGF-21, preferentially expressed in the liver

We isolated cDNA encoding a novel FGF (210 amino acids) from mouse embryos. As this is the 21st documented FGF, we tentatively term it FGF-21. FGF-21 has a hydrophobic amino terminus ( approximately 30 amino acids), which is a typical signal sequence, and appears to be a secreted protein. The expression of FGF-21 mRNA in mouse adult tissues was examined by Northern blotting analysis. FGF-21 mRNA was most abundantly expressed in the liver, and also expressed in the thymus at lower levels. We also isolated human cDNA encoding FGF-21 (209 amino acids). Human FGF-21 is highly identical ( approximately 75% amino acid identity) to mouse FGF-21. Among human FGF family members, FGF-21 is most similar ( approximately 35% amino acid identity) to FGF-19.     

A chicken mRNA similar to heterogeneous nuclear ribonucleoprotein H1

Heterogeneous nuclear ribonucleoproteins are predominantly nuclear RNA-binding proteins that function in a variety of cellular activities. The objective of these experiments was to clone a cDNA for a chicken protein similar to other previously reported heterogeneous ribonucleoproteins for other species. The 5' and 3' ends of the chicken mRNA were cloned using Rapid Amplification of cDNA Ends (RACE). Subsequently, the expression of the mRNA sequence was confirmed via Northern analysis. The deduced amino acid sequence was approximately 86% identical to corresponding regions of human, mouse, or zebrafish proteins similar to heterogeneous nuclear ribonucleoprotein H1. The expression data confirmed the size of the predicted mRNA sequence. The newly identified sequence may be employed in future studies aimed at understanding the role of heterogeneous nuclear ribonucleoproteins in avian species.     

Isolation of a cDNA encoding the adenovirus E1A enhancer binding protein: a new human member of the ets oncogene family.

The cDNA encoding adenovirus E1A enhancer-binding protein E1A-F was isolated by screening a HeLa cell lambda gt11 expression library for E1A-F site-specific DNA binding. One cDNA clone produced recombinant E1A-F protein with the same DNA binding specificity as that endogenous to HeLa cells. Sequence analysis of the cDNA showed homology with the ETS-domain, a region required for sequence-specific DNA binding and common to all ets oncogene members. Analysis of the longest cDNA revealed about a 94% identity in amino acids between human E1A-F and mouse PEA3 (polyomavirus enhancer activator 3), a recently characterized ets oncogene member. E1A-F was encoded by a 2.5kb mRNA in HeLa cells, which was found to increase during the early period of adenovirus infection. In contrast, ets-2 mRNA was significantly reduced in infected HeLa cells. The results indicate that E1A enhancer binding protein E1A-F is a member of the ets oncogene family and is probably a human homologue of mouse PEA3.