A novel ras-related gene family

We have identified a new family of ras genes, the rho genes, which share several properties with the more classical ras gene family consisting of N-, K-, and H-ras. The rho genes, first isolated from a cDNA library from the abdominal ganglia of Aplysia, encode proteins that share 35% amino acid homology with H-ras. Evolutionarily conserved counterparts of rho have been detected in yeast, in Drosophila, in rat, and in man. Sequence analysis reveals over 85% homology between the human and Aplysia proteins. The ras and rho gene products share several common properties; both are 21,000 daltons, both reveal C-terminal sequences required for membrane attachment, and both show blocks of strong internal homology, suggesting that the two proteins may share common functions but may use these functions in different ways.     

Cloning, sequence analysis and expression of a cDNA encoding a novel insulin-like growth factor binding protein (IGFBP-2).

Insulin-like growth factors bind with high affinity to specific binding proteins in extracellular fluids. To identify structural characteristics of IGF-binding proteins that might define their physiological roles, we determined the complete primary structure of a novel human IGF-binding protein (IGFBP-2) from a cloned cDNA. The cDNA encodes a 328 amino acid IGF-binding protein precursor which contains a 39-residue signal peptide. The mature 289 amino acid IGFBP-2 has a predicted Mr of 31,325. Chinese hamster ovary (CHO) cells stably transformed with the IGFBP-2 cDNA secreted a 36 kd protein which bound, with different affinities, IGFII and IGFI, but did not bind insulin. The predicted protein sequence of this IGF-binding protein shares extensive amino acid homology (greater than 85%) with the IGF-binding protein secreted by rat BRL-3A cells, but less than 40% homology with human IGFBP-1. Therefore IGFBP-2, and not IGFBP-1 as previously suggested, represents the human homologue of the rat BRL-BP (alpha IGFBP-2). Moreover, from alignment of the predicted protein sequences of IGFBP-1 and IGFBP-2, extensive conservation of the distribution of cysteine residues is observed. Although the overall amino acid homology shared by these proteins is not high, we suggest that they represent a family of structurally related human IGFBPs. Southern blot analysis of human DNA demonstrates that IGFBP-2 is encoded by a single-copy gene, different from that of IGFBP-1.     

Expansion of the complement receptor gene family. Identification in the mouse of two new genes related to the CR1 and CR2 gene family

Human cDNA probes encoding the C3b/C4b complement receptor, CR1, have been used to identify, in the mouse, two new genes which are related to CR1 but which appear to encode a different protein product. These new mouse genes, arbitrarily designated mouse genes X and Y, hybridize specifically to three different cDNA probes derived from human CR1. The degree of hybridization homology between the mouse X and Y genes suggests they are very closely related to one another; however, the chromosomal localization of the mouse X gene to chromosome 8 and the mouse Y gene to chromosome 1 indicates they are distinct gene sequences. The mRNA species detected with the X and/or Y (X/Y) sequences are approximately 2000 bases in length, but vary in both quantity and size depending upon the tissue analyzed. DNA sequence analysis of a cDNA specific for the X and Y sequences indicates the mature protein(s) will contain the 60 amino acid consensus repeat characteristic of a group of other proteins including CR1, the C3d receptor (CR2), H, C4 binding protein (C4bp), the interleukin 2 (Il 2) receptor and others. The identity of the mouse X and Y genes, and the function of the proteins which they encode, is not known; however, the small size of the mRNA and the tissue specific expression suggests they do not encode mouse CR1 or CR2 but instead encode a related protein (or proteins) which is expressed in a wide variety of mouse tissues.     

Cloning, sequence analysis and transcriptional expression of a ras gene of the edible basidiomycete Lentinus edodes

In the edible basidiomycete, Lentinus edodes, the presence of a high level of intracellular cyclic AMP (cAMP) is closely related to the onset of fruiting and/or primordium formation. Since a close relationship between intracellular cAMP levels and expression of ras genes was reported for organisms such as Saccharomyces cerevisiae and Dictyostelium discoideum, we have cloned and sequences a ras gene homologue from L. edodes (Le.), and analyzed its expression during development of the fungus. This gene, named Le.ras, has a coding capacity of 217 amino acids (aa) interrupted by six small introns. The deduced Le.Ras protein exhibited the highest homology to the Schizosaccharomyces pombe RAS protein (219 aa): 86% homology in the N-terminal 80-aa sequence and 74% homology in the next 80 aa. The Le.ras gene was transcribed at similar levels during mycelial development in fruiting-body formation, suggesting no direct correlation of Le.ras expression with intracellular cAMP levels in this organism.     

Three different genes in S. cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase

We have isolated three genes (TPK1, TPK2, and TPK3) from the yeast S. cerevisiae that encode the catalytic subunits of the cAMP-dependent protein kinase. Gene disruption experiments demonstrated that no two of the three genes are essential by themselves but at least one TPK gene is required for a cell to grow normally. Comparison of the predicted amino acid sequences of the TPK genes indicates conserved and variable domains. The carboxy-terminal 320 amino acid residues have more than 75% homology to each other and more than 50% homology to the bovine catalytic subunit. The amino-terminal regions show no homology to each other and are heterogeneous in length. The TPK1 gene carried on a multicopy plasmid can suppress both a temperature-sensitive ras2 gene and adenylate cyclase gene.     

白毛黑眼兔眼部虹膜Trp1、Trp2基因克隆与分析

目的 克隆日本大耳白兔白毛黑眼系(白毛黑眼兔)眼部虹膜Trp1、Trp2基因,获取其完整的外显子序列.进一步推测这两个基因编码的蛋白,并分析其特征.方法 从白毛黑眼兔的黑色虹膜组织中提取RNA,并反转录成cDNA.利用来自小鼠、大鼠和人的同源引物,扩增获得白毛黑眼兔Trp1、Trp2基因外显子片段.然后对已知片段进行3' RACE和5'RACE,从而获得白毛黑眼兔Trp1、Trp2基因外显子完整序列.利用相关软件对获得序列进行翻译和分析.结果 首次获得了白毛黑眼兔Trp1、Trp2基因外显子全序列.该实验兔Trp1基因的编码序列全长1604个碱基,其核苷酸序列与人的相似度为87.9％,与小鼠的相似度为82.7％.TRP1成熟蛋白包含513氨基酸,氨基酸序列与人的相似度为89.8％,与小鼠的相似度为86.6％.该实验兔Trp2基因序列全长1554个碱基,其核苷酸序列与人的相似度为83.2％,与小鼠的相似度为81.9％.TRP2成熟蛋白包含494个氨基酸,其序列与人的一致度为84.2％,与小鼠的一致度为84.4％.结论 本研究获得的TRP1、TRP2的序列与已知的家兔酪氨酸相关蛋白家族成员TYR的序列进行比对,结果显示这三种蛋白之间有较高的相似度,并且TRP1和TRP2蛋白序列表现出了酪氨酸酶家族结构上的保守性和特有的结构特征.     

Evolutionary and structural relationships among the group-specific component, albumin and alpha-fetoprotein.

The group-specific component (Gc) is a plasma protein that binds vitamin D. Recent characterization of human Gc cDNA demonstrated homology with serum albumin and alpha-fetoprotein. This study compares the sequences of the three proteins and demonstrates a strong evolutionary relationship. Albumin, alpha-fetoprotein and Gc evolved from an ancestral gene containing an intragenic triplication. Comparison of the amino acid sequences and patterns of double disulfide bonds suggests that the Gc gene may have diverged from an ancestral gene earlier in evolution than the genes encoding albumin and alpha-fetoprotein. Analysis of the amino acid and nucleotide sequences of the three internal domains of Gc revealed 19-23% amino acid sequence identity and the localization of three homology blocks with 40-44% nucleotide sequence identity. The deduced amino sequence of Gc furnished data for comparing its molecular configuration based on the predicted secondary structure with those predicted for human albumin and alpha-fetoprotein. Utilization of Gc cDNA has also led to the identification of its genomic DNA and detection of a human DNA polymorphism.     

The human Rab genes encode a family of GTP-binding proteins related to yeast YPT1 and SEC4 products involved in secretion

Seven cDNA clones corresponding to the rab1, rab2, rab3A, rab3B, rab4, rab5, and rab6 genes were isolated from a human pheochromocytoma cDNA library. They encode 23-25 kDa polypeptides which share approximately 30-50% homology and belong to the ras superfamily. The rab1, rab2, rab3A, and rab4 proteins are the human counterparts of the rat rab gene products that we have previously characterized. Comparison of the seven human rab proteins with the yeast YPT1 (YPT1p) and SEC4 (SEC4p) proteins reveals highly significant sequence similarities. H-rab1p shows 75% amino acid identity with YPT1p and may be therefore considered as its human counterpart. The other proteins share approximately 40% homology with YPT1p and SEC4p. The homology (approximately 30%) between these rab proteins and p21ras is restricted to the four conserved domains involved in the GTP/GDP binding. Human rab proteins were produced in Escherichia coli. Large amounts of rab proteins in soluble form can be extracted and purified without the use of detergents. All six proteins bind GTP and exhibit GTPase activities. A possible involvement of the rab proteins in secretion is discussed.     

Yeast RNC1 encodes a chimeric protein, RhoNUC, with a human rho motif and deoxyribonuclease activity.

The yeast Saccharomyces cerevisiae contains an endoexonuclease yNucR that has been implicated in both recombination and repair. We describe the isolation and characterization of the corresponding gene. Within the predicted N-terminal half of the protein there is extensive homology (approximately 50%) with human rho genes, which are related to the ras oncogene, particularly in the proposed GTP-binding region. The C-terminal region, which is related to the Escherichia coli recC protein, presumably encodes the endoexonuclease activity. The yNucR may thus represent a new class of GTP-binding proteins. Because of the chimeric nature of the polypeptide, this protein is renamed RhoNUC (rather than the original yNucR) and the gene is RNC1 for Rho-associated-NuClease. Over expression of the gene leads to altered cell growth and nuclear morphology. We propose that the gene plays an important role in cell development as well as DNA repair/recombination.     

Analysis of the genomic organisation of a small chromosome of Leishmania braziliensis M2903 reveals two genes encoding GTP-binding proteins, one of which belongs to a new G-protein family and is an antigen

Leishmania braziliensis M2903 contains a highly amplified small chromosome. This work is aimed at resolving its structural organization and determining whether this unusual chromosome contains specific genes encoding proteins with important functions in disease pathology or drug resistance. Our results show that the M2903 250-kb small chromosome contains LD1 sequences and has an inverted repeat structure. The LD1 sequences and two cDNAs (cDNA2 and cDNA53) were mapped on a cosmid contig, and the two cDNAs and the corresponding genomic fragments from the small chromosome were sequenced. The gene encoding cDNA2 predicts a putative GTP-binding protein with homology to other GTP-binding proteins only in the G-1 domain region; however, four other conserved motifs can be recognized. Sequence similarity to cDNA53 is located in at least five chromosomes, and its small chromosome copy is a pseudogene. An open reading frame downstream of the cDNA53 pseudogene predicts another GTP-binding protein that belongs to a new G-protein family with an unusual conserved GTP-binding domain and a newly characterized conserved sequence motif. A portion of this GTP-binding protein gene was studied previously in L. aethiopica as a recombinant antigen that reacts with human antibodies.     

Cloning of Japanese flounder Paralichthys olivaceus CD3 cDNA and gene, and analysis of its expression

Two distinct CD3 homologue cDNAs, CD3-1 and CD3-2, were isolated from a Japanese flounder leukocyte cDNA library. CD3-1 consisted of 961 bp encoding 178 amino acid residues, and CD3-2 consisted of 927 bp encoding 182 amino acid residues. The two deduced amino acid sequences had an identity of 95.1%, and neither had N-linked glycosylation sites. The identities between the Japanese flounder CD3s and previously reported CD3s (CD3 epsilon, CD3 gamma, or CD3 delta) of Xenopus laevis, chicken, and various mammals were approximately 25%. The Japanese flounder CD3s had an extracellular domain, a CXXCXE motif, and an immunoreceptor tyrosine-based activation motif (ITAM), each of which are important characteristics of CD3 chains. Furthermore, the positions of four cysteine residues in the extracellular domain were preserved in both of the Japanese flounder CD3s. A phylogenetic tree based on the amino acid sequences confirmed that the Japanese flounder CD3s are closer to CD3 epsilon than to CD3 gamma and CD3 delta. However, the gene structure of Japanese flounder CD3 is identical to the chicken and Xenopus CD3 gamma/delta genes and the mammalian CD3 delta gene. Southern blot hybridization and the DNA sequence of the CD3 gene of homocloned Japanese flounder indicated that the CD3 gene exists as a single copy. Southern blot hybridization also showed the presence of a polymorphic variant of Japanese flounder CD3. An RT-PCR analysis detected Japanese flounder CD3 mRNA in several organs that contained lymphocytes. The proportion of CD3-positive cells in the peripheral blood leukocytes was 34.9%.     

A pore-forming protein,perforin, from a non-mammalian organism,Japanese flounder,<Emphasis Type="Italic"> Paralichthys olivaceus</Emphasis>

A perforin cDNA of Japanese flounder, Paralichthys olivaceus, was cloned from a cDNA library of kidney stimulated with ConA/PMA. The full-length cDNA is 2,157 bp, which encodes 587 amino acids. The Japanese flounder perforin gene consists of five exons and four introns, with a length of approximately 3 kb. The amino acid sequence of the Japanese flounder perforin is 36% identical to that of rat perforin and 37% identical to amino acid sequences of mouse and human perforin. The Japanese flounder perforin also showed low homology to human and mouse complement components (C6, C7, C8 and C9), ranging from 19% to 24%. However, the membrane attack complex/perforin domain is conserved. A phylogenetic analysis placed the Japanese flounder perforin in the same cluster with other known mammalian perforins. RT-PCR analysis revealed that the perforin gene was expressed in the peripheral blood leukocytes, head kidney, trunk kidney, spleen, heart, gill and intestine of healthy fish. Recombinant perforin produced in insect cells using the baculovirus expression system showed calcium-dependent hemolytic activity.     

伴花生球蛋白cDNA克隆及其在花生发育种子中的表达

利用伴花生球蛋白多克隆抗体,免疫筛选花生品种汕油523成熟子叶中期cDNA文库得到6个阳性克隆.经过DNA序列测定和同源性分析确定为2组(Ahyα和Ahyβ) ,2组序列之间的同源性为97%.Ahyβ与花生过敏原Ara h1 p17以及Ahyα与花生过敏原Ara h1p41b的核苷酸相同性达到99%以上.以Ahy-βcDNA为探针的Northern blot分析结果表明,伴花生球蛋白基因在发育的花生种子中大量表达,而在幼苗的叶片中不表达.对成熟中期花生子叶表达序列标签(EST)分析,获得了包括5种花生球蛋白、2种伴花生球蛋白、6种conglutin蛋白的EST共70条,占总转录本的17%.     

Human and mouse LSP1 genes code for highly conserved phosphoproteins

With use of the mouse LSP1 cDNA we isolated a human homologue of the mouse LSP1 gene from a human CTL cDNA library. The predicted protein sequence of human LSP1 is compared with the predicted mouse LSP1 protein sequence and regions of homology are identified in order to predict structural features of the LSP1 protein that might be important for its function. Both the human and mouse LSP1 proteins consist of two domains, an N-terminal acidic domain and a C-terminal basic domain. The C-terminal domains of the mouse and human LSP1 proteins are highly conserved and include several conserved, putative serine/threonine phosphorylation sites. Immunoprecipitation of LSP1 protein from 32P-orthophosphate-loaded cells show that both the mouse and human LSP1 proteins are phosphoproteins. The sequences of the putative Ca2(+)-binding sites present in the N-terminal domain of the mouse LSP1 protein are not conserved in the human LSP1 protein; however, a different Ca2(+)-binding site may exist in the human protein, indicating a functional conservation rather than a strict sequence conservation of the two proteins. The expression of the human LSP1 gene follows the same pattern as the expression of the mouse LSP1 gene. Southern analysis of human genomic DNA shows multiple LSP1-related fragments of varying intensity in contrast to the simple pattern found after similar analysis of mouse genomic DNA. By using different parts of the human LSP1 cDNA as a probe, we show that most of these multiple bands contain sequences homologous to the conserved C-terminal region of the LSP1 cDNA. This suggests that there are several LSP1-related genes present in the human genome.     

Studies on hst, a transforming gene which belongs to a new superfamily of growth factors

The hst gene was originally identified in surgically obtained human gastric mucosae as a transforming gene which could transform NIH3T3 cells morphologically. The hst cDNA clone was synthesized from mRNA of one of the NIH3T3 transformants. A human leukocyte genomic library was screened with this cDNA clone, and an hst genomic fragment was obtained. This genomic fragment itself had transforming activity, and the protein coding sequences were proved to be completely identical to those of the cDNA clone prepared from mRNA of the NIH3T3 transformant. This fact suggests that rearrangement or other structural alterations in the coding sequence are not required for the activation of the hst gene. The predicted hst protein consists of 206 amino acids and has a significant homology (40-50%) to fibroblast growth factors and int-2 protein. They together make up a new superfamily of growth factors and transforming genes.     

Generation of 919 expressed sequence tags from immature flower buds and gene expression analysis using expressed sequence tags in the model plant Lotus japonicus

Lotus japonicus has received increased attention as a potential model legume plant. In order to study gene expression in reproductive organs and to identify genes that play a crucial function in sexual reproduction, we constructed a cDNA library from immature flower buds containing anthers at the stage of developing tapetum cells in L. japonicus, and characterized 919 expressed sequence tags (ESTs) randomly selected from a cDNA library of the immature flower buds. The 919 ESTs analyzed were clustered into 821 non-redundant EST groups. As a result of a database search, 436 groups (53%) out of the 821 groups showed sequence similarity to genes registered in the public database. Out of these 436 groups, 109 groups showed similarity to genes encoding hypothetical proteins whose function had not yet been estimated. Three hundred eighty five groups (47%) showed no significant homology to known sequences and were classified as novel sequences. A comparison of 821 non-redundant EST sequences and EST sequences derived from the whole plant L. japonicus revealed that 474 EST sequences derived from immature flower buds were not found in the EST sequences of the whole plant. In order to confirm the expression pattern of potential reproductive-organ specific EST clones, nine clones, which were not matched to ESTs derived from the whole plant, were selected, and RT-PCR analysis was performed on these clones. As a result of RT-PCR, we found two novel anther specific clones. One clone was homologous to a gene encoding human cleft lip and palate associated transmembrane protein (CLPTM1) like protein, and the other clone did not show a significant similarity to any genes deposited in the public database. These results indicate that ESTs analyzed here represent a valuable resource for finding reproductive-organ specific genes in Lotus japonicus.