Plant cytosolic ribosomal protein S11 and chloroplast ribosomal protein CS17. Their primary structures and evolutionary relationships

We have isolated cDNA clones specific for Arabidopsis thaliana cytosolic ribosomal protein S11 and plastid ribosomal protein CS17, both of which are encoded in the nuclear genome, through the use of the corresponding soybean and pea cDNAs as probes, respectively. The nucleotide sequences of all four cDNAs were determined. The amino acid sequences derived from these cDNA sequences show that the soybean and A. thaliana S11 cDNAs encode proteins that are homologous to rat ribosomal protein S11 and that the pea and A. thaliana CS17 cDNAs encode proteins that are homologous to Escherichia coli ribosomal protein S17. The plant S11 cytosolic ribosomal proteins also show significant sequence similarity to both E. coli ribosomal protein S17 and plastid CS17 indicating that these are all related proteins. Comparison of A. thaliana CS17 with A. thaliana S11 and with E. coli S17 suggests that CS17 is more related to S17 than it is to S11. These results support the idea that the gene encoding CS17 was derived from a prokaryotic endosymbiont and not from a duplication of the eukaryotic S11 gene.     

Mapping of 12 bovine ribosomal protein genes using a bovine radiation hybrid panel

Twelve bovine ribosomal protein genes, for which sequence data had been acquired from complementary deoxyribonucleic acid (cDNA) clones isolated from a cattle skin cDNA library, were mapped. As ribosomal protein genes are a group of highly conserved house keeping genes, specific primers were designed to span the intron-exon splice sites and to amplify intronic sequences, in order to obtain bovine-specific polymerase chain reaction (PCR) products. Two of 12 ribosomal protein genes were genotyped in this way and the remaining 10 were mapped using additional primers designed from within the intron. Eleven previously unmapped ribosomal protein genes were localized and one previously reported ribosomal protein gene localization was confirmed. The 12 ribosomal protein genes mapped in this study are spread over 10 chromosomes, including the X chromosome. The locations show conservation of comparative map position in cattle and human.     

Isolation of a large number of novel mammalian genes by a differential cDNA library screening strategy.

As part of the ongoing human and mouse genome projects, the aim of this study was to isolate novel, previously uncharacterized, genes from mouse testis. Two approaches were compared for their effectiveness in isolating novel genes: random, vs differential, complementary DNA (cDNA) cloning methods. In the differential approach, only the cDNA clones containing rare sequences (as determined by preliminary clone hybridization) are further analyzed; in the random approach, cDNA clones are isolated at random from the cDNA library. More than two hundred cDNA clones altogether were analyzed, using a PCR-mediated amplification and sequencing strategy. A comparison of these sequences to nucleic acid and protein sequence databases, revealed that 84% of the isolated rare cDNA clones represented new, previously uncharacterized mouse genes. In contrast, less than 63% of the cDNA clones isolated at random from cDNA libraries, contained novel genes. Thus, the probability of isolating new, previously uncharacterized, mammalian genes from cDNA libraries can be markedly improved by focusing efforts on clones containing rare sequences.     

cDNA cloning and characterization of interleukin 2-induced genes in a cloned T helper lymphocyte

Clonal expansion of antigen-specific lymphocytes is an important aspect of the immune response. Interleukin 2 (IL2) is largely responsible for the amplification of antigen-specific T cells. In this study, the changes in gene expression accompanying interleukin 2 stimulation of T cells are examined, using a cloned T helper lymphocyte line as a model system. To isolate cDNA clones of IL2-induced genes, a cDNA library was screened by differential hybridization. Twenty-one different cDNA clones were isolated by this method, comprising six glycolytic enzymes, vimentin, alpha-tubulin, beta-actin, gamma-actin, ERp99, elongation factor 2, ribosomal phosphoprotein P1, the DNA-binding protein dbpB/YB-1, as well as seven clones which do not correspond to any previously described sequences. These clones are used to study the time course of expression and the sensitivity to cycloheximide inhibition of IL2-induced mRNAs. In addition, the tissue specificity of the unidentified mRNAs is examined, and two of these are shown to be expressed at high levels in normal mouse brain, with much lower or undetectable levels in the other tissues tested. These cDNA clones will be useful in future studies to determine the molecular basis of IL2-induced gene expression.     

Rat ribosomal protein L35a multigene family: molecular structure and characterization of three L35a-related pseudogenes

The rat ribosomal protein L35a gene comprises a multigene family which contains 15-20 members as shown by the Southern blot analysis using L35a cDNA as a probe. We isolated 15 independent clones which contained distinct genes from a rat genomic library. Analysis of the restriction sites showed that all of them lacked the intervening sequences. Thermal stability of the hybrid molecules between these genes and the cDNA indicated that the similarity of the genes to the cDNA sequence varied. The nucleotide sequences of three genes gRL35a-A, gRL35a-B and gRL35a-G were determined. They shared some characteristics; namely: they lacked the intervening sequences, they contained (A)-rich tracts, and they were flanked by direct repeats. Two genes, gRL35a-A and gRL35a-B, contained a sequence completely identical to that of the cDNA. The nucleotide sequence of the 5' flanking region of gRL35a-B showed a significant homology with that of the same region of mouse ribosomal protein L32-related unmutated processed genes. Although this region of gRL35a-B contained the sequences homologous to the TATA box and the CCAAT box, gRL35a-B was not transcribed in an in vitro assay system. Thus, the L35a gene family comprises mostly processed pseudogenes. Further, Southern blot analysis in various animals indicated that the multigene construction of this ribosomal protein gene was a feature of mammalian genes. The origin and the evolutionary aspect of processed pseudogenes are discussed.     

黄鳝性腺高表达的核糖体蛋白基因

采用高密点阵技术从黄鳝雄性性腺cDNA文库中获得8个克隆,序列分析和BLAST结果显示它们编码的蛋白质分别与40S核糖体蛋白S4,S9,S16,S17,S20和60S核糖体蛋白L7, L18a,L29高度同源。 根据黄鳝RP蛋白序列和其他物种的相应同源序列构建ML系统发生树,显示核糖体蛋白基因在进化中高度保守。核糖体蛋白基因不仅可作为分子进化分析的有利工具,而且从它们的表达模式显示RP基因除具有看家基因的功能外,很有可能参与包括性腺分化等过程的发育调控。Abstract: 8 cDNA clones have been isolated from a cDNA library prepared from swamp eel testies by macroarray. DNA sequence analysis and database search showed that they encode 8 proteins which are highly homologous to 40S ribosomal proteins S4,S9,S16,S17,S20 and 60S riobosomal proteins L7, L18a,L29. Phylogenetic trees (ML) based on ribosomal protein genes from swamp eel and other organisms has been reconstructed, which showed that ribosomal protein genes were highly conserved during evolution. These results suggested that ribosomal protein genes as house keeping genes may play roles in developmental regulation such as sexual differentiation and can also be used as markers for the study of molecular evolution.     

Isolation and characterization of cDNA clones for chicken major histocompatibility complex class II molecules

The chicken major histocompatibility complex (MHC), the B complex, is being intensively analysed at the DNA level. To further probe the molecular structure of chicken MHC class II genes, cDNA clones coding for chicken MHC class II (B-L) p chain molecules were isolated from an inbred G-B2 Leghorn chicken spleen and liver. Twenty-nine cDNA clones were isolated from the spleen and eight cDNA clones were isolated from the liver. Based on restriction maps, most clones could be clustered into one family of genes. Four cDNA clones were sequenced (S7, S10 and S19 from the spleen and L1, which was identical to S19, from the liver). Complete amino acid sequences of B-Lβ chain molecules were predicted from the nucleotide sequences of the cDNA clones. Although both the nature and the location of the conserved residues were similar in chicken and mammalian sequences, some species-specific differences were found, suggesting that the structures of the B-L molecules of this haplotype are similar, but not identical, to their mammalian counterparts.     

Ly-49 multigene family. New members of a superfamily of type II membrane proteins with lectin-like domains

Ly-49 (YE1/48, A1) is a dimer protein expressed on subpopulations of murine NK cells. It is a member of a superfamily of type II transmembrane proteins containing carbohydrate recognition domains (CRD). In the mouse genome, the detection of multiple restriction fragments that cross-hybridize with Ly-49 cDNA probes suggests the presence of related genes. In this study, we have isolated several genomic clones encoding portions of CRD sequences highly homologous to the CRD of Ly-49. By using primers based on the consensus sequences of the genomic clones, expression of Ly-49-related genes was detected by the polymerase chain reaction in various organs, including lung, kidney, liver, spleen, and thymus. Two full-length cDNA clones that are highly homologous to the Ly-49 gene were subsequently isolated from a lung cDNA library. At the nucleotide level, the two clones are 72% and 80% identical to Ly-49 in their translated regions, but their sequences are different from those of the genomic clones characterized to date. The two cDNA clones potentially encode type II transmembrane proteins containing CRD that are very similar to Ly-49. These amino acid sequences are also homologous to other members of the superfamily of CRD-containing type II transmembrane proteins, including hepatic lectins and the low affinity IgER (CD23). The homology is most evident in the CRD but is also significant in other domains. These results demonstrate the existence of several functional genes that are highly related to Ly-49. These genes comprise a subfamily within the superfamily of type II transmembrane proteins containing CRD.     

病毒感染草鱼胸腺的EST分析和免疫相关基因的鉴定

以感染草鱼(Ctenopharyngodon idellus)出血病病毒(GCHV)的草鱼胸腺为材料,构建了草鱼胸腺的SMARTcDNA文库.筛选文库获得到1933条有效EST序列.BLASTX分析显示,583条序列在公共数据库中能找到同源基因(E-value≤1.00E 10-3,Identities≥30%),另外1350条序列则找不到显著同源性.已知基因按具体功能可划分为6类,大部分与细胞内的各种生理过程、细胞结构以及免疫防御相关.研究结果从分子水平上表明鱼类的胸腺在机体感染病毒的免疫反应中发挥重要作用,同时也表明胸腺组织在病毒感染后可能表达很多目前还不清楚功能的新基因.     

Ribosomal protein L2 in Saccharomyces cerevisiae is homologous to ribosomal protein L1 in Xenopus laevis. Isolation and characterization of the genes

By cross-hybridization with a cDNA probe for the Xenopus laevis ribosomal protein L1 we have been able to isolate the homologous genes from a Saccharomyces cerevisiae genomic library. We have shown that these genes code for a ribosomal protein which was previously named L2. In yeast, like in X. laevis, these genes are present in two copies per haploid genome and, unlike the vertebrate counterpart, they do not contain introns. Amino acid comparison of the X. laevis L1 and S. cerevisiae L2 proteins has shown the presence of a highly conserved protein domain embedded in very divergent sequences. Although these sequences are very poorly homologous, they confer an overall secondary structure and folding highly conserved in the two species.     

Large scale gene expression analysis of CBA/J mouse strain fetal thymus using cDNA-array hybridizations

The CBA/J inbred mouse strain constitutes an interesting in vivo model-system for studies on molecular genetics of thymus ontogeny. Using RT-PCR method we have found previously that several immune system related genes as interleukins and MHC are differentially expressed. During this period the onset of T-cell receptor beta rearrangements also occur. To know which other genes are modulated during the ontogeny of the thymus, the mRNA expression levels of fetal thymus (15 and 16 days gestation) of CBA/J mouse strain were measured by hybridization with a set of four macroarrays containing a panel of 6,144 IMAGE cDNA clones from MTB thymus library. We found 145 differentially expressed sequences; 44 were up- and 101 down-regulated in the thymus at 15-16 days gestation. Among these sequences, only 20 are identified as genes whose functions are known and 125 are still unknown. Our data demonstrated that, despite intense research on maturation of the immune system focusing on the activity of several well-characterized genes, the large scale expression profile during thymus ontogeny is still an open matter. The use of cDNA-array technology is an affordable method to identify new genes that may play a role in this phenomenon.     

Large scale gene expression analysis of CBA/J mouse strain fetal thymus using cDNA-array hybridizations

The CBA/J inbred mouse strain constitutes an interesting in vivo model-system for studies on molecular genetics of thymus ontogeny. Using RT-PCR method we have found previously that several immune system related genes as interleukins and MHC are differentially expressed. During this period the onset of T-cell receptor beta rearrangements also occur. To know which other genes are modulated during the ontogeny of the thymus, the mRNA expression levels of fetal thymus (15 and 16 days gestation) of CBA/J mouse strain were measured by hybridization with a set of four macroarrays containing a panel of 6,144 IMAGE cDNA clones from MTB thymus library. We found 145 differentially expressed sequences; 44 were up- and 101 down-regulated in the thymus at 15–16 days gestation. Among these sequences, only 20 are identified as genes whose functions are known and 125 are still unknown. Our data demonstrated that, despite intense research on maturation of the immune system focusing on the activity of several well-characterized genes, the large scale expression profile during thymus ontogeny is still an open matter. The use of cDNA-array technology is an affordable method to identify new genes that may play a role in this phenomenon.     

Cloning of a Leishmania major gene encoding for an antigen with extensive homology to ribosomal protein S3a

Following purification by affinity chromatography, a Leishmania major S-hexylglutathione- binding protein of molecular mass 66kDa was isolated. The immune serum against the parasite 66kDa polypeptide when used to screen a L. major cDNA library could identify clones encoding for the human v-fos transformation effector homologue, namely ribosomal protein S3a, and thus was named LmS3a-related protein (LmS3arp). A 1027bp cDNA fragment was found to contain the entire parasite gene encoding for a highly basic protein of 30kDa calculated molecular mass sharing homology to various ribosomal S3a proteins from different species. Using computer methods for a multiple alignment and sequence motif search, we found that LmS3arp shares a sequence homology to class theta glutathione S-transferase mainly in a segment containing critical residues involved in glutathione binding. These new findings are discussed in the light of recent published data showing multiple function(s) of the ribosomal proteins S3a.     

Molecular cloning and analysis of cDNA sequences for two ribosomal proteins from Artemia. The coordinate expression of genes for ribosomal proteins and elongation factor 1 during embryogenesis of Artemia

The large subunit of eukaryotic ribosomes contains acidic phosphoproteins which are related to L7/L12 from Escherichia coli. In the brine shrimp Artemia these proteins are designated eL12 and eL12'. We have isolated cDNA clones for these proteins from a cDNA bank that was constructed by the use of size-fractionated poly(A)-rich RNA (8-10S fraction) from Artemia and a synthetic oligonucleotide as primer. Clones containing DNA sequences coding for eL12 and eL12 were characterized by hybrid-selected translation and DNA sequencing. The proteins eL12 and eL12' share an identical peptide of 22 amino acids at their carboxy termini whereas the remaining part of the protein shows little sequence homology. The nucleotide sequences show a different codon use for the amino acids in the common carboxy terminus, thereby excluding a common exon coding for this part of both proteins. Despite the differences in amino acid sequence in the major part of eL12 and eL12' the proteins have a considerable degree of homology on the basis of the distribution of hydrophobic and hydrophilic amino acids over the polypeptide chains, in agreement with a related folding and function of both proteins. Relative levels of mRNA coding for eL12, eL12' and elongation factor 1 alpha were determined during the development of Artemia from a dormant cyst to a nauplius. The data show a coordinate expression of the genes for EF-1 alpha and both ribosomal proteins, excluding a differential expression of the genes for these related ribosomal proteins during embryogenesis. Analysis of the gene copy number for eL12 and eL12' indicates the presence of a few genes for each protein.     

Molecular cloning of transcripts that accumulate during the late G1 phase in cultured mouse cells

To identify previously undetected genes that might be involved in later stages of the transition from a quiescent state (G0) to the DNA synthetic phase (S) of murine cells, we set out to isolate cDNA clones derived from mRNAs that appear late in G1 phase in serum-stimulated cells. A lambda-cDNA library was prepared using poly(A)+ RNA from chemically transformed Balb/c 3T3 cells (BP/A31) that had been brought to quiescence and subsequently stimulated for 12 h with serum. From the first screening of approximately 21,000 recombinant phage plaques, about 100 clones were isolated that hybridized to a single-stranded cDNA pool derived from stimulated-cell RNA but not to DNAs made from resting-cell RNA. Eventually, six different clones were identified. The mRNAs from five of these genes increased gradually during the G0 to S transition, in contrast to the "immediate-early" rise of c-myc mRNA or the later rise of thymidine kinase mRNA. These six clones were sequenced and compared to the GenBank database. Clones LG-80, LG-2, and LG-69 are highly homologous to beta-actin, lactate dehydrogenase, and alpha-tubulin. Clones LG-5, LG-61, and LG-74 had no significant homologies to known sequences. A subtractive cDNA library was used to isolate two additional clones, Sub-S1 and Sub-S2; these have homologies to enolase and ribosomal protein L32. Additional studies that examine the function and regulation of these newly identified "late response" genes in the pre-DNA synthesis pathway are in progress.     

Prediction of the coding sequences of mouse homologues of KIAA gene: IV. The complete nucleotide sequences of 500 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a mouse cDNA project to predict protein-coding sequences of mouse homologues of human KIAA and FLJ genes since 2001. As an extension of these projects, we herein present the entire sequences of 500 mKIAA cDNA clones and 4 novel cDNA clones that were incidentally identified during this project. We have isolated cDNA clones from the size-fractionated mouse cDNA libraries derived from 7 tissues and 3 types of cultured cells. The average size of the 504 cDNA sequences reached 4.3 kb and that of the deduced amino acid sequences from these cDNAs was 807 amino acid residues. We assigned the integrity of CDSs from the comparison with the corresponding human KIAA cDNA sequences. The comparison of mouse and human sequences revealed that two different human KIAA cDNAs are derived from single genes. Furthermore, 3 out of 4 proteins encoded in the novel cDNA clones showed moderate sequence similarity with human KIAA proteins, thus we could obtain new members of KIAA protein families through our mouse cDNA projects.