Sequence and chromosomal location of the I-309 gene. Relationship to genes encoding a family of inflammatory cytokines

We previously reported the isolation and characterization of a cDNA clone, I-309, that encodes a small secreted protein produced by activated human T lymphocytes. This protein is structurally related to a large number of recently identified proteins that are secreted upon cellular activation. In this report we describe the isolation and characterization of the gene encoding I-309. The genomic organization is essentially identical to that found in the genes encoding the structurally similar proteins TCA-3, hJE/MCP-1, and mJE, strengthening the hypothesis that these genes are evolutionarily related. The region of the I-309 gene 5' of the mRNA cap site exhibits extensive nucleotide sequence homology with the same region of the murine gene TCA-3, providing additional evidence that I-309 and TCA-3 are likely to be homologs. Finally, panels of rodent-human somatic cell hybrids were used to map the I-309 gene to human chromosome 17. In conjunction with recent mapping data from other laboratories, this result suggests the presence of a cluster of related genes on this chromosome.     

Selection of Arabidopsis genes encoding secreted and plasma membrane proteins

Secreted and plasma membrane proteins play crucial roles in a variety of physiological and developmental processes of multicellular organisms. Systematic cloning of the genes encoding these proteins is therefore of general interest. An effective method of trapping signal sequences was first described by Tashiro et al. (1993), and a similar yet more efficient method was reported by Klein et al. (1996) and Jacobs et al. (1997). In this study, we carried out the latter yeast-based signal sequence trap to clone genes from Arabidopsis thaliana encoding secreted and plasma membrane proteins. Of 144 sequenced cDNA clones, 18% are identical to previously cloned Arabidopsis thaliana genes, 12% are homologous to genes identified from various organisms, and 46% are novel. All of the isolated genes identical or homologous to previously reported genes are either secreted or plasma membrane proteins, and the remaining novel genes appear to contain functional signal sequences based on computer-aided sequence analysis. The full-length cDNA clones of one homologous gene and another novel gene were isolated and sequenced. The deduced amino acid sequences suggest that the former encodes a secreted protein, and the latter encodes a type 1 membrane protein. These results indicate that the signal sequence trap method is effective and useful for the isolation of plant genes encoding secreted and plasma membrane proteins.     

Isolation, characterization, and UV-stimulated expression of two families of genes encoding polypeptides of related structure in human epidermal keratinocytes.

By screening of a cDNA library made on mRNA isolated from UV-irradiated human epidermal keratinocytes for sequences whose relative concentration increases in the cytoplasm after irradiation, we have isolated 40 cDNA clones (T. Kartasova, B. J. C. Cornelissen, P. Belt, and P. van de Putte, Nucleic Acids Res. 15:5945-5962, 1987). Here we describe two distinct groups of cDNA clones which do not cross-hybridize to each other but nevertheless encode proteins of very similar primary structure. These polypeptides are small (8 to 10 kilodaltons) and exceptionally rich in proline, cysteine, and glutamine and have similar repeating elements not found elsewhere. The new proteins were designated sprI and sprII (small, proline rich). The presence of prolines and cysteines suggests that they may be either structural proteins with a strong secondary structure or metal-binding proteins such as metallothioneins. Southern blot and sequence analyses of the cDNAs indicate that at least the sprII group of clones represents a family of related genes. The nucleotide sequence of both groups seems to be conserved upon evolution. The level of mRNAs corresponding to the two groups of cDNAs is increased in the cytoplasm of human epidermal keratinocytes after both UV irradiation and treatment with 4-nitroquinoline 1-oxide or 12-O-tetradecanoylphorbol 13-acetate.     

A family of small inducible proteins secreted by leukocytes are members of a new superfamily that includes leukocyte and fibroblast-derived inflammatory agents, growth factors, and indicators of various activation processes

We have isolated and characterized four cDNA clones that encode mRNA expressed more abundantly in Con A-activated mouse helper T cells than by resting T cells. One mRNA encoded a approximately 14-kDa protein with a hydrophobic N-terminal sequence and was abundantly expressed by the Th 2 subset of Th cells, but was not expressed by Th 1 cells. The remaining three mRNA encoded related approximately 8-kDa secreted proteins that are part of a family of small, secreted, and inducible mouse and human proteins. This family of proteins is itself distantly related to another family of growth and inflammatory factors that are associated with various lymphoid and fibroblast activation phenomena. One of the small, inducible, secreted proteins has a predicted mature N terminus identical to that of the previously described macrophage inflammatory protein.     

DNA Sequence Analysis of a Complementary DNA for Cold-Regulated Arabidopsis Gene cor15 and Characterization of the COR 15 Polypeptide

Previous studies have indicated that changes in gene expression occur in Arabidopsis thaliana L. (Heyn) during cold acclimation and that certain of the cor (cold-regulated) genes encode polypeptides that share the unusual property of remaining soluble upon boiling in aqueous solution. Here, we identify a cDNA clone for a cold-regulated gene encoding one of the “boiling-stable” polypeptides, COR15. DNA sequence analysis indicated that the gene, designated cor15, encodes a 14.7-kilodalton hydrophilic polypeptide having an N-terminal amino acid sequence that closely resembles transit peptides that target proteins to the stromal compartment of chloroplasts. Immunological studies indicated that COR15 is processed in vivo and that the mature polypeptide, COR 15m, is present in the soluble fraction of chloroplasts. Possible functions of COR 15m are discussed.     

Organization and expression of two tandemly oriented genes encoding ribulosebisphosphate carboxylase/oxygenase activase in barley

We have isolated and structurally characterized genomic DNA and cDNA sequences encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rbu-P2 carboxylase) activase from barley (Hordeum vulgare L.). Three Rbu-P2 carboxylase activase (Rca) polypeptides are encoded in the barley genome by two closely linked, tandemly oriented nuclear genes (RcaA and RcaB); cDNAs encoding each of the three Rbu-P2 carboxylase activase polypeptides were isolated from cDNA libraries of barley leaf mRNA. RcaA produces two mRNAs, which encode polypeptides of 42 and 46 kDa, by an alternative splicing mechanism identical to that previously reported for spinach and Arabidopsis Rca genes (Werneke, J.M., Chatfield, J.M., and Ogren, W. L. (1989) Plant Cell 1, 815-825). RcaB is transcribed to produce a single mRNA, which encodes a mature peptide of 42 kDa. Genomic Southern blots indicate that RcaA and RcaB represent the entire Rbu-P2 carboxylase activase gene family in barley. The genes share 80% nucleotide sequence identity, and the 42-kDa polypeptides encoded by RcaA and RcaB share 87% amino acid sequence identity. Coding regions of the two barley Rca genes are separated by 1 kilobase pair of flanking DNA. DNA sequence motifs similar to those thought to control light-regulated gene expression in other nuclear-encoded plastid polypeptide genes are found at the 5' end of both barley Rca genes. Probes specific to three mRNAs were used to determine the relative contribution each species makes to the total Rca mRNA pool.     

Group 1 CD1 genes in rabbit

CD1 is an Ag-presenting molecule that can present lipids and glycolipids to T cells. The CD1 genes were first identified in the human, and since then, homologs have been identified in every mammalian species examined to date. Over a decade ago, CD1B and CD1D homologs were identified in the rabbit. We have extended this earlier study by identifying additional CD1 genes with the goal of developing the rabbit as an animal model to study the function of CD1 proteins. We constructed a thymocyte cDNA library and screened the library with CD1-specific probes. Based on nucleotide sequence analyses of the CD1(+) cDNA clones obtained from the library, we have identified two CD1A genes and one CD1E gene as well as determined the complete sequence of the previously identified CD1B gene. The CD1E(+) cDNA clones lacked the transmembrane and cytoplasmic domains and, if translated, would encode for a soluble or secreted CD1E protein. In addition, expression studies demonstrated that the CD1 genes were expressed in peripheral lymphoid tissues as well as in skin, gut, and lung. Of interest is the finding that CD1A2, CD1B, and CD1E genes were found to be expressed by rabbit B cell populations. The rabbit, with a complex CD1 locus composed of at least two CD1A genes, one CD1B gene, one CD1D gene, and one CD1E gene, is an excellent candidate as an animal model to study CD1 proteins.     

Molecular cloning of cDNAs derived from a novel human intestinal mucin gene

A human small intestinal lambda gt11 cDNA library was screened with antibodies to deglycosylated small intestinal mucin. Four partial cDNA clones were isolated that define a novel human mucin gene. These include two partial cDNA clones, SIB 124 and SIB 139, that contain 51 nucleotide tandem repeats which encode a seventeen amino acid repetitive peptide with a consensus sequence of HSTPSFTSSITTTETTS. SIB 139 hybridized to messages produced by small intestine, colon, colonic tumors and also by high mucin variant LS174T colon cancer cells. The gene from which cDNAs SIB 124 and SIB 139 are derived (proposed name MUC 3) maps to chromosome 7, distinct from other known human mucin genes.     

Mitogenic activation of human T cells induces two closely related genes which share structural similarities with a new family of secreted factors

Previously we have isolated about 60 novel cDNA clones whose corresponding mRNAs are induced by mitogenic activation in human peripheral blood T cells. Here we describe the primary structure and regulation of two such cloned genes, pAT 464 and pAT 744, which may encode new lymphokines/cytokines. Similar to IL-2, both genes require the synergy of agents such as PHA and PMA for optimal expression, and, in addition, the induction of both is sensitive to the immunosuppressive drug cyclosporin A. The two genes can be expressed in T cells, B cells, and the promyelocytic cell line HL60, but they are not expressed in human fibroblasts, suggesting that their expression is restricted to hematopoietic lineages. The predicted peptides encoded by these two clones feature hydrophobic N-terminal leaders characteristic of secreted proteins. The predicted size of both proteins is about 8 kDa upon cleavage of the putative leader peptide. pAT 464 and pAT 744 are very similar to each other and also share some critical amino acid similarity with a newly emerging family of secreted factors including connective tissue activating factor III, platelet factor 4, an IFN-gamma-induced factor, macrophage inflammatory protein, and a factor chemotactic to neutrophils (3-10C, monocyte-derived neutrophil chemotactic factor, neutrophil-activating factor). Some of these factors have been shown to display functions associated with an inflammatory response and/or have mitogenic activities. Collectively, the data presented here suggest that pAT 464 and pAT 744 encode novel lymphokines/cytokines which may play roles during an immune response similar to those enacted by these structurally related factors.     

Molecular cloning of actin genes in Trichomonas vaginalis and phylogeny inferred from actin sequences

The parasitic protozoan Trichomonas vaginalis is known to contain the ubiquitous and highly conserved protein actin. A genomic library and a cDNA library have been screened to identify and clone the actin gene(s) of T. vaginalis. The nucleotide sequence of one gene and its flanking regions have been determined. The open reading frame encodes a protein of 376 amino acids. The sequence is not interrupted by any introns and the promoter could be represented by a 10 bp motif close to a consensus motif also found upstream of most sequenced T. vaginalis genes. The five different clones isolated from the cDNA library have similar sequences and encode three actin proteins differing only by one or two amino acids. A phylogenetic analysis of 31 actin sequences by distance matrix and parsimony methods, using centractin as outgroup, gives congruent trees with Parabasala branching above Diplomonadida.     

Identification of novel homologues of three low molecular weight subunits of the mitochondrial bc1 complex

Large-scale random cDNA sequencing projects have been started for several organisms and are a valuable tool for the analysis of quantitative and qualitative aspects of gene expression. However, the reliability of the obtained data is limited as most of the clones are only partially analysed on one strand. As a consequence the sequence entries derived from random cDNA sequencing projects usually comprise incomplete open reading frames. They nevertheless define complete and reliable coding sequences, if two prerequisites are fullfilled: (i) the clones encode very small proteins, and (ii) the clones have a high frequency in the cDNA-banks. The present study describes the use of cDNA databases for the identification of homologues of three low-molecular-weight subunits of the mitochondrial bc₁ complex, termed the QCR6, QCR9 and QCR10 proteins. These polypeptides are only characterized for a small number of organisms, have a scarcely defined function and exhibit a low degree of structural conservation if compared between different species. Several clones were identified for each polypeptide by searches with TBLASTN using the known sequences as probes. Most of the database entries contain complete open reading frames and sequencing queries could be excluded due to the abundancy of the clones. Multiple sequence alignments are presented for all three polypeptides and consensus sequences are given which may provide a basis for the investigation of the proteins by site-directed mutagenesis.     

Nucleotide sequences of two pea cDNA clones encoding the small subunit of ribulose 1,5-bisphosphate carboxylase and the major chlorophyll a/b-binding thylakoid polypeptide

Two major chloroplast proteins are encoded by nuclear genes and synthesized on free cytoplasmic ribosomes: the small subunit of ribulose 1,5-bisphosphate carboxylase and the apoprotein components of the chlorophyll a/b light harvesting complex. We have recently reported the isolation of two cDNA clones from pea which encode both the small subunit of ribulose 1,5-bisphosphate carboxylase (pSS15) and the polypeptide 15 (pAB96), the major chlorophyll a/b binding protein (Broglie, R., Bellemare, G., Bartlett, S., Chua, N.-H., and Cashmore, A. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 7304-7308). To further characterize these clones, we determined their nucleotide sequence. Clone pSS15 contains a 691-base pair cDNA insert which encodes the entire 123 amino acids of the mature small subunit protein. In addition, this clone also encodes 33 amino acids of the NH2-terminal transit peptide extension and 148 nucleotides of the 3' noncoding region preceding the poly(A)tail. A second cDNA clone (pAB96) contains an 833-nucleotide insert which encodes most of polypeptide 15. The DNA sequence of this cloned cDNA was used to deduce the previously undetermined amino acid sequence of this integral thylakoid membrane protein. The nucleotide sequence of the cDNA clone, pSS15, should provide information concerning the role of the transit sequence in the transport of cytoplasmically synthesized chloroplast proteins. Similarly, the deduced amino acid sequence of polypeptide 15 will provide information for predicting its orientation in thylakoid membranes as well as its role in binding chlorophyll.     

Receptor-like protein kinase genes of Arabidopsis thaliana

The isolation of a maize cDNA clone that encodes a membrane spanning protein kinase related to the self-incompatibility glycoproteins (SLG) of Brassica and structurally similar to the growth factor receptor tyrosine kinases has recently been reported. Three distinct receptor-like protein kinase (RLK) cDNA clones from Arabidopsis thaliana have now been identified. Two of the Arabidopsis RLK genes encode SLG-related protein kinases but have different patterns of expression: one is expressed predominantly in rosettes while the other is expressed primarily in roots. The third RLK gene contains an extracellular domain that consists of 21 leucine-rich repeats that are analogous to the leucine-rich repeats found in proteins from humans, flies and yeast. The Arabidopsis leucine-rich gene is expressed at equivalent levels in roots and rosettes. These results show that there are several genes in higher plants that encode members of the receptor protein kinase superfamily. The structural diversity and differential expression of these genes suggest that each plays a distinct and possibly important role in cellular signaling in plants.     

Analysis of cDNA sequences from mouse testis

Few mammalian proteins involved in chromosome structure and function during meiosis have been characterized. As an approach to identify such proteins, cDNA clones expressed in mouse testis were analyzed by sequencing and Northern blotting. Various cDNA library screening methods were used to obtain the clones. First, hybridization with cDNA from testis or brain allowed selection of either negative or differentially expressed plaques. Second, positive plaques were identified by screening with polyclonal antisera to prepubertal testis nuclear proteins. Most clones were selected by negative hybridization to correspond to a low abundance class of mRNAs. A PCR-based solid-phase DNA sequencing protocol was used to rapidly obtain 306 single-pass cDNA sequences totaling more than 104 kb. Comparison with nucleic acid and protein databases showed that 56% of the clones have no significant match to any previously identified sequence. Northern blots indicate that many of these novel clones are testis-enriched in their expression. Further evidence that the screening strategies were appropriate is that a high proportion of the clones which do have a match encode testisenriched or meiosis-specific genes, including the mouse homolog of a rat gene that encodes a synaptonemal complex protein.The nucleotide sequence data reported in this paper have been submitted to Genbank and have been assigned the accession numbers L26606–1.26848.     

Divergent genes for translation initiation factor eIF-4A are coordinately expressed in tobacco.

Three cDNA clones coding for eukaryotic translation initiation factor 4A, eIF-4A, were isolated from a Nicotiana plumbaginifolia root cDNA library by heterologous screening. The clones comprise two distinct gene classes as two clones are highly similar while the third is divergent. The genes belong to a highly conserved gene family, the DEAD box supergene family, although the divergent clone contains a DESD box rather than the characteristic DEAD box. The two clones are representatives of separate small multigene families in both N. plumbaginifolia and N. tabacum. Representatives of each family are coordinately expressed in all plant organs examined. The 47 kD polypeptide product of one clone, overexpressed in E. coli, crossreacts immunologically with a rabbit reticulocyte eIF-4A polyclonal antibody. Taken together the data suggest that the two Nicotiana eIF-4A genes encode translation initiation factors. The sequence divergence and the coordinate expression of the two Nicotiana eIF-4A families provide an excellent system to determine if functionally distinct eIF-4A polypeptides are required for translation initiation in plants.     

Equine infectious anemia virus tat: insights into the structure, function, and evolution of lentivirus trans-activator proteins.

Equine infectious anemia virus (EIAV) contains a tat gene which is closely related to the trans-activator genes of the human and simian immunodeficiency viruses. Nucleotide sequence analysis of EIAV cDNA clones revealed that the tat mRNA is composed of three exons; the first two encode Tat and the third may encode a Rev protein. Interestingly, EIAV Tat translation is initiated at a non-AUG codon in exon 1 of the mRNA, perhaps allowing an additional level of gene regulation. The deduced amino acid sequence of EIAV tat, combined with functional analyses of tat cDNAs in transfected cells, has provided some unique insights into the domain structure of Tat. EIAV Tat has a C-terminal basic domain and a highly conserved 16-amino-acid core domain, but not the cysteine-rich region, that are present in the primate immunodeficiency virus Tat proteins. Thus, EIAV encodes a relatively simple version of this kind of trans activator.