Integrated genetic and physical map of the 1q31-->q32.1 region, encompassing the RP12 locus, the F13B and HF1 genes, and the EEF1AL11 and RPL30 pseudogenes.

The gene for autosomal recessive retinitis pigmentosa (RP12) with preserved para-arteriolar retinal pigment epithelium was previously mapped close to the F13B gene in region 1q31-->q32.1. A 4-Mb yeast artificial chromosome contig spanning this interval was constructed to facilitate cloning of the RP12 gene. The contig comprises 25 sequence-tagged sites, polymorphic markers, and single-copy probes, including five newly obtained probes. The contig orders the F13B and HF1 genes, as well as five expressed sequence tags, with respect to the integrated genetic map of this region. Homozygosity mapping resulted in refinement of the candidate gene locus for RP12 to a 1. 3-cM region. Currently, approximately 1 Mb of the contig is represented in P1-derived artificial chromosome (PAC) clones. Direct screening of a cDNA library derived from neural retina with PACs resulted in identification of the human elongation factor 1alpha pseudogene (EEF1AL11) and a human ribosomal protein L30 pseudogene (RPL30). A physical and genetic map covering the entire RP12 candidate gene region was constructed.     

A complete map of the human ribosomal protein genes: assignment of 80 genes to the cytogenetic map and implications for human disorders

Mapping of the human ribosomal protein (RP) genes has been completed, and all 80 different genes were placed on a cytogenetic map of the human genome. Because of the existence of processed pseudogenes, the localization of the RP genes was complicated, and five genes had remained to be mapped. Here we developed a novel strategy to identify sequence-tagged sites (STSs) at introns of the RP genes, and we localized RPL14, RPL22, RPL35, RPL36, and RPL39 within the chromosomes by radiation hybrid mapping. Unlike the case of eubacteria or archaebacteria, human RP genes are widely scattered about the genome. Together with the previous results, both sex chromosomes and 20 autosomes (all but chromosomes 7 and 21) were found to carry one or more RP genes. To explore the possible involvement of RP genes in human disorders, all 80 genes were assigned to cytogenetic bands according to a published cytogenetic BAC-STS map of the human genome. We compared the assigned positions with candidate regions for Mendelian disorders and found certain genes that might be involved in particular human disorders.     

Identification and expression of an autosomal paralogue of ribosomal protein S4, X-linked,in mice: Potential involvement of testis-specific ribosomal proteins in translation and spermatogenesis

Recent studies have revealed heterogeneity in the structure of eukaryotic cytoplasmic ribosomes, including a difference in protein composition. It has been proposed that this heterogeneity, or the specialized ribosome, contributes to tissue development and homeostasis through selective mRNA translation, although this remains largely unclear. Our previous proteomic survey of rodent ribosomes found the testis-specific ribosomal proteins L10-like and L39-like, which are paralogues of the X-linked ribosomal proteins L10 and L39, respectively. We have hypothesized that the rodent testis provides a good model for examining the possible functional importance of ribosome heterogeneity. In the present study, a new paralogue of X-linked ribosomal protein S4 has been identified in the mouse testis. The gene encoding this paralogue was autosomal, intronless and expressed predominantly in the testis. It appeared that this paralogue was included in polysomes as a component of the ribosome. Although these properties were similar to those of the ribosomal proteins L10-like and L39-like, this S4 paralogue and L10-like showed partially different expression patterns in spermatogenic cells. These findings are discussed in relation to the unique evolution of genes encoding a paralogue of ribosomal protein S4 in mammals and to the significance of testis-specific paralogues of ribosomal proteins in active ribosomes during spermatogenesis.     

Molecular evolutionary analyses of the Arabidopsis L7 ribosomal protein gene family

Cytoplasmic ribosomal protein (r-protein) genes in Arabidopsis thaliana are encoded by 80 multigene families that contain between two and seven members. Gene family members are typically similar at the protein sequence level, with the most divergent members of any gene family retaining 94% identity, on average. However, three Arabidopsis r-protein families - S15a, L7 and P2 - contain highly divergent family members. Here, we investigated the organization, structure, expression and molecular evolution of the L7 r-protein family. Phylogenetic analyses showed that L7 r-protein gene family members constitute two distinct phylogenetic groups. The first group including RPL7B, RPL7C and RPL7D has homologs in plants, animals and fungi. The second group represented by RPL7A is found in plants but has no orthologs from other fully-sequenced eukaryotic genomes. These two groups may have derived from a duplication event prior to the divergence of animals and plants. All four L7 r-protein genes are expressed and all exhibit a differential expression in inflorescence and flowers. RPL7A and RPL7B are less expressed than the other genes in all tissues analyzed. Molecular characterization of nucleic and protein sequences of L7 r-protein genes and analysis of their codon usage did not indicate any functional divergence. The probable evolution of an extra-ribosomal function of group 2 genes is discussed.     

牧草盲蝽实时定量PCR内参基因的筛选

【目的】筛选出适合牧草盲蝽Lygus pratensis在不同条件下稳定表达的内参基因。【方法】选取编码牧草盲蝽α-微管蛋白、β-微管蛋白、肌动蛋白、延伸因子、琥珀酸脱氢酶复合体A、泛素、转录起始因子TFIID亚基、谷胱甘肽S-转移酶、核糖体蛋白L32及TATA盒结合蛋白的10条基因为候选内参基因,采用qRT-PCR技术测定其在牧草盲蝽不同性别成虫、成虫不同组织、不同龄期、对功夫菊酯不同抗性成虫、不同温度及不同杀虫剂分别处理后的成虫共6类样品中的表达量;采用BestKeeper, geNorm, NormFinder及RefFinder 4种计算程序对各候选基因的表达稳定性进行评价。【结果】依据获得的有效qRT-PCR数据前提,利用不同计算方法综合分析得出：在牧草盲蝽成虫不同组织中和对功夫菊酯不同抗性成虫中最稳定表达的内参基因均为RPL32;不同温度处理、不同性别成虫中、不同杀虫剂处理和不同龄期牧草盲蝽中最稳定表达的内参基因分别为UBQ, SDHA, β-tubulin和TAF。通过geNorm软件判断配对变异数确定的内参基因最佳数目,结合RefFinder对基因表达稳定性综合排序的结果可得：牧草盲蝽不同成虫组织中、不同性别成虫中、不同龄期、对功夫菊酯不同抗性成虫中、不同温度及杀虫剂分别处理的成虫等各组的最优内参基因组合分别为RPL32+TAF, SDHA+GST, TAF+GST+UBQ, RPL32+GST, UBQ+ACT+β-tubulin和β-tubulin+TAF+RPL32。【结论】本研究获得的牧草盲蝽在不同条件下表达最稳定及最适内参基因组合,都可用于后续的基因定量表达研究。     

Primary structure of dihydrofolate reductase and mitochondrial ribosomal protein L36 genes from the basidiomycete Coprinus cinereus.

We amplified and sequenced the dihydrofolate reductase (DHFR) gene of the basidiomycete Coprinus cinereus. Downstream of the DHFR coding region, a mitochondrial (mt) ribosomal protein L36 (RPL36) gene was discovered in the opposite orientation to DHFR gene. Putative polyadenylation signals of the two genes overlapped, both containing the 8-bp palindrome 5'-aatatatt-3'. The finding that C. cinereus DHFR gene is closely clustered with a mt protein gene strongly suggests that C. cinereus DHFR is closely related to mt function and evolution. The amino acid sequence of C. cinereus DHFR is most homologous to eukaryotic proteins such as Cryptococcus neoformans and Pneumocystis carinii DHFRs. However, the sequence of C. cinereus mt RPL36 closely resembles RPL36 of bacteria and cyanobacteria such as Synechocystis sp. and Escherichia coli. This result strongly supports the serial endosymbiotic theory of the development of ancestral eukaryotes, and suggests that C. cinereus mt RPL36 gene originated from the ancestral eubacterial genome.     

Differential expression of house-keeping genes of Aspergillus nidulans during sexual development

The rpl3 gene and the rpl37 gene for Aspergillus nidulans ribosomal protein L3 (RPL3) and RPL37, which were identified as located on chromosome I and chromosome III, respectively, were isolated from chromosome-specific cosmid libraries. The nucleotide sequences of both of the rpl3 gene and the rpl37 gene identified the ORFs of 392 amino acids and 92 amino acids, respectively. Both of the two genes were present in a single copy. The expression of both genes together with two other house-keeping genes, the rps16 gene for RPS16 and the gene for gamma-actin, was analyzed during sexual development. All four genes showed nearly identical expression patterns in that each gene expression reached its maximum after 2 h, decreased thereafter, and increased again after 30-40 h of induction of sexual development.     

Bt毒素诱导下小菜蛾实时定量PCR 内参基因的筛选

【目的】筛选出Bt毒素诱导后的小菜蛾Plutella xylostella (L.)的实时定量PCR最适内参基因。【方法】选取核糖体18S rRNA (18S rRNA)、 肌动蛋白（ACTB）、 延伸因子（EF1）、3-磷酸甘油醛脱氢酶（GAPDH）、 核糖体蛋白L32 (RPL32)、 核糖体蛋白S13 （RPS13）、 核糖体蛋白S20 （RPS20）和β-微管蛋白(TUB)基因作为候选内参基因, 以geNorm、 Normfinder和BestKeeper软件分析这8个基因在Bt毒素诱导后的小菜蛾不同品系中肠组织中的表达稳定性。并应用筛选出来的内参基因分析小菜蛾氨肽酶2（aminopeptidase N2, APN2）基因的表达水平。【结果】geNorm软件以RPS13和EF1为最稳定内参基因, NormFinder和BestKeeper软件均以RPS13和RPL32为最稳定基因。使用3种不同内参基因分析Bt毒素诱导后的小菜蛾Bt抗性和敏感品系中ANP2表达水平时, 新的内参基因EF1和传统内参基因RPL32表现了良好的稳定性, 二者作为标准化因子, ANP2表达量结果基本一致, 而使用18S rRNA作为内参基因, 却导致部分表达量分析结果有所误差。【结论】筛选出PRS13,RPL32和EF1可以作为小菜蛾某些试验条件下的内参基因, 对小菜蛾基因表达研究奠定了一定基础, 也对其他昆虫内参基因的筛选具有参考价值。     

The human large subunit ribosomal protein L36A-like contacts the CCA end of P-site bound tRNA

Periodate-oxidized tRNA (tRNAox), the 2′,3′-dialdehyde derivative of tRNA, was used as a zero-length active site-directed affinity labeling reagent, to covalently label proteins at the binding site for the 3′-end of tRNA on human 80S ribosomes. When human 80S ribosomes were reacted with tRNA^Aspox positioned at the P-site, in the presence of an appropriate 12 mer mRNA, a set of two tRNAox-labeled ribosomal proteins (rPs) was observed. The majorily labeled protein was identified as the large subunit rP L36a-like (RPL36AL) by means of mass spectrometry. Intact tRNA^Asp competed with tRNA^Aspox for the binding to the P-site, by preventing tRNA-protein cross-linking with RPL36AL. Altogether, the data presented in this report are consistent with the presence of RPL36AL at or near the binding site for the CCA end of the tRNA substrate positioned at the P-site of human 80S ribosomes. It is the first time that a ribosomal protein is found in an intimate contact (i.e. at a zero-distance) with a nucleotide of the conserved CCA terminus of P-site tRNA which is the substrate of peptidyl transferase reaction. RPL36AL which is strongly conserved in eukaryotes belongs to the L44e family of rPs, a representative of which is Haloarcula marismortui RPL44e.     

Identification and characterization of the gene for Drosophila L3 ribosomal protein

A cDNA clone that encodes a Drosophila homologue of ribosomal protein L3 was isolated from a Drosophila ovary gridded cDNA library. The Drosophila ribosomal protein L3 gene (RpL3) is highly conserved with ribosomal protein L3 genes in other organisms. It is a single copy gene and maps to position 86D5–10 on polytene chromosomes. A Minute gene in this region, M(3)86D, is a possible candidate to encode RPL3. RPL3 message is expressed ubiquitously. A partial RPL8 cDNA clone was also isolated and mapped to 62F.     

Annotation pattern of ESTs from Spodoptera frugiperda Sf9 cells and analysis of the ribosomal protein genes reveal insect-specific features and unexpectedly low codon usage bias

MOTIVATION: A whole set of Expressed Sequence Tags (ESTs) from the Sf9 cell line of Spodoptera frugiperda is presented here for the first time. By this way we want to identify both conserved and specific genes of this pest species. We also expect from this analysis to find a class of protein sequences providing a tool to explore genomic features and phylogeny of Lepidoptera. RESULTS: The ESTs display both housekeeping as well as developmentally regulated genes, and a high percentage of sequences with unknown function. Among the identified ORFs, almost all ribosomal proteins (RPs) were found with high EST redundancy and hence sequence accuracy. The codon usage found among RP genes is in average surprisingly much less biased in Lepidoptera than in other organisms. Other Spodoptera genes also displayed a low bias, suggesting a general genome expression feature in this Lepidoptera. We also found that the L35A and L36 RP sequences, respectively, display 40 and 10 amino-acid insertions, both being present only in insects. Sequence analysis suggests that they are probably not subjected to a strong selective pressure and may be good phylogenetic markers for Lepidoptera. Most interestingly, the Lepidoptera sequences of 9 RP genes displayed a specific signature different from the canonical one. We conclude that the RP family allows valuable comparative genomics and phylogeny of Lepidoptera. AVAILABILITY: All EST sequence data are available from the private 'Spodo-Base' upon request.     

Structural comparison of yeast ribosomal protein genes.

The primary structure of the genes encoding the yeast ribosomal proteins L17a and L25 was determined, as well as the positions of the 5'- and 3'-termini of the corresponding mRNAs. Comparison of the gene sequences to those obtained for various other yeast ribosomal protein genes revealed several similarities. In all split genes the intron is located near the 5'-side of the amino acid coding region. Among the introns a clear pattern of sequence conservation can be observed. In particular the intron-exon boundaries and a region close to the 3'-splice site show sequence homology. Conserved sequences were also found in the leader and trailer regions of the ribosomal protein mRNAs. The 5'-flanking regions of the yeast ribosomal protein genes appeared to contain sequence elements that many but not all ribosomal protein genes have in common, and therefore may be implicated in the coordinate expression of these genes. The amino acid coding sequences of the ribosomal protein genes show a biased codon usage. Like most yeast ribosomal protein molecules, L17a and L25 are particularly basic at their N-terminus.