Cloning of a novel gene encoding human thioredoxin-like protein

mRNA differential display (DDRT-PCR) has been used to analyze different human fetal brain tissues of different developmental stages (13- and 33-week). According to the sequence of one EST obtained in this assay, a pair of primers have been designed to screen the arrayed human fetal brain cDNA library. A1 .2-kb cDNA clone has been found. This cDNA consists of an 867 bp open reading frame, a 132 bp 5' untranslated sequence and a 209 bp 3' untranslated sequence with a typical polyadenylation signal. The coding region predicts a protein of 289 amino acids. Its N-terminal of 105 residues is highly homologous to human thioredoxin, while no homology has been found in the databases with its C-terminal of 184 residues. Its N-terminal region also contains the conserved active site sequence CGPC (Cys-Gly-Pro-Cys) of thioredoxin. It was named human Thioredoxin-like gene (hTRXL).     

A Drosophila Minute gene encodes a ribosomal protein

Minute genes have long constituted a special problem in Drosophila genetics. For at least 50-60 different genes scattered throughout the genome, dominant mutations and/or deficiencies have been recognized which result in a common phenotype consisting of short thin bristles, slow development, reduced viability, rough eyes, small body size and etched tergites. Schultz proposed that the Minute loci encode similar but separate functions involved in growth and division common to all cells. Atwood and Ritossa suggested that Minute loci encode components of the protein synthetic machinery, specifically the transfer RNA genes; this now seems unlikely on grounds of both mapping and mutability studies. More recently, we and others suggested that the Minute loci are ribosomal protein genes. We report here that transformation with a cloned 3.3-kilobase (kb) region containing the gene encoding the large subunit ribosomal protein 49 (rp49) suppresses the dominant phenotypes of Minute (3)99D, a previously undescribed Minute associated with a chromosomal deficiency of the 99D interval. This activity is specific to the 99D Minute as it does not suppress other Minute loci elsewhere in the genome. This result provides direct evidence that the Minute locus at the 99D interval encodes the ribosomal protein 49.     

一个与油菜黄化相关的未知功能基因克隆及表达

以构建的油菜幼叶黄化突变体Cr3529消减文库中一个未知功能的差异表达基因片段为基础,应用RACE-PCR技术,扩增并克隆到两个cDNA全序列,分别命名为BnCr4和Bn-Cr4-1.测序结果显示,BnCr4编码区序列大小为1395 bp,编码465个氨基酸,而BnCr4-1编码区序列长1311 bp,编码437个氨基酸.BLAST结果表明它们与拟南芥中的一个未知功能基因的cDNA同源性分别为87%和80%.功能预测显示它们含有与蛋白质的修饰作用有关的多个活性位点,如磷酸化、糖基化、酰胺化以及磷酸泛酰巯基乙胺结合位点,可能是一种新的与cAMP介导的蛋白质磷酸化与去磷酸化作用有关的蛋白.Northen杂交结果显示该基因在Cr3529子叶期和幼叶期的表达高于野生型油菜,显示该基因的表达与突变性状紧密相关.最后,原核表达了BnCr4,得到了与预计分子量相同的融合蛋白.     

Isolation ofosRACD gene encoding a small GTP-binding protein from rice

Using an improved version of mRNA differential display technology, we have obtained a differentially displayed fragment RDP-8. Homologous comparison indicated that the fragment RDP-8 has high homology with the gene encoding maize small GTP-binding protein. By screening cDNA library of the rice Nongken 58N pan icle using the newly obtained fragment RDP-8 as probe, we further found the full-length cDNA of osRACD gene that encodes a rice small GTP-binding protein. Asco mpared with maize RACD gene, the osRACD of rice shows remarkable homology in both nucleotide sequence and amino acid sequence, 88% and 97% respectively. Evidence from RT-PCR study indicates that osRACD gene is related to photoperiod fertility conversion of photoperiod sensitive genic male sterility (PSGMS) rice.     

Amplification of a gene encoding a p53-associated protein in human sarcomas.

Despite extensive data linking mutations in the p53 gene to human tumorigenesis, little is known about the cellular regulators and mediators of p53 function. MDM2 is a strong candidate for one such cellular protein; the MDM2 gene was originally identified by virtue of its amplification in a spontaneously transformed derivative of mouse BALB/c cells and the MDM2 protein subsequently shown to bind to p53 in rat cells transfected with p53 genes. To determine whether MDM2 plays a role in human cancer, we have cloned the human MDM2 gene. Here we show that recombinant-derived human MDM2 protein binds human p53 in vitro, and we use MDM2 clones to localize the human MDM2 gene to chromosome 12q13-14. Because this chromosomal position appears to be altered in many sarcomas, we looked for changes in human MDM2 in such cancers. The gene was amplified in over a third of 47 sarcomas, including common bone and soft tissue forms. These results are consistent with the hypothesis that MDM2 binds to p53, and that amplification of MDM2 in sarcomas leads to escape from p53-regulated growth control. This mechanism of tumorigenesis parallels that for virally-induced tumours, in which viral oncogene products bind to and functionally inactivate p53.     

Cloning of a down-regulated gene encoding small GTP binding protein in hybrid wheat

Previous studies showed that differential gene expression between wheathybrids and their parents was responsible for the heterosis. To provide an insight into the molecular basis of wheat heterosis, one cDNA, designated TaRab, was identified from the cDNA library of wheat seedling leaves. The sequence comparison in GenBank revealed that TaRab is homologous to a group of genes encoding Rab-GTP binding protein. Semi-quantitative RT-PCR analysis indicated that TaRab was expressed in all plant tissues examined, but at slightly higher level in leaves. Further analysis exhibited that TaRab displayed lower expression in hybrid than in its patents in both roots and leaves, which was in agreement with the original results of suppression subtractive hybridization. TaRab was located on chromosome 7B and C-7DS5-0.36 by in silico mapping. The relationship between differential expression of TaRab and the molecular basis of wheat heterosis was also discussed.     

Randomization of protein encoding gene during species evolution

Employing chi-square teat, the authors observed the bias of 4 kinds of bases’ locating in 3 codon positions in gene sequence, and proved that there is a positive correlation between higher chi-square value and protein coding structure. By this, the observatlon that genes undergo progressive reduction in chi-values along evolution direction will reflect the progressive randomization of gene structures and the loss of the characteristics of protein coding sequences. The result proposed that during the evolution of life, genes are unexpectedly degenerating.     

Cloning and sequence analysis of a gene encoding polygalacturonase-inhibiting protein from cotton

Polygalacturonase-inhibiting proteins (PGIP) play important roles in plant defense of pathogen, especially fungi. A pair of degenerated primers is designed based on the conserved sequence of 20 other known pgip genes and used to amplify Gossypium barbadense cultivation 7124 cDNA library by touch-down PCR. A 561 bp internal fragment of the pgip gene is obtained and used to design the primers for rapid amplification of cDNA ends. A composite pgip gene sequence is constructed from the products of 5′ and 3′ RACE, which are 666 bp and 906 bp respectively. Analysis of nucleic acid sequence shows 69.2% and 68.7% similarity to Citrus and Poncirus pgip genes, respectively. Its open reading frame of the gene encodes a polypeptide of 330 amino acids, in which 10 leucine-rich repeats arrange tandemly. A new set of primers is designed to the 5′ and 3′ ends of the gene, which allows amplification of the full-length gene from the cotton cDNA library. Genomic DNA analysis reveals that this gene has no intron.     

The yeast ubiquitin gene: head-to-tail repeats encoding a polyubiquitin precursor protein

Ubiquitin, a 76-residue protein, occurs in cells either free or covalently joined to a variety of protein species, from chromosomal histones to cytoplasmic proteins. Conjugation of ubiquitin to proteolytic substrates is essential for the selective degradation of intracellular proteins in higher eukaryotes. We show here that a protein homologous to human ubiquitin exists in the yeast Saccharomyces cerevisiae, and that yeast extracts conjugate human ubiquitin to a variety of endogenous proteins in an ATP-dependent reaction. We have isolated the S. cerevisiae ubiquitin gene and found it to contain six consecutive ubiquitin-coding repeats in a found it to contain six consecutive ubiquitin-coding repeats in a head-to-tail arrangement. This apparently unique gene organization suggests that yeast ubiquitin is generated by processing of a precursor protein in which several exact repeats of the ubiquitin amino acid sequence are joined directly via Gly-Met peptide bonds between the last and first residues of mature ubiquitin, respectively. Ubiquitin-coding yeast DNA repeats are restricted to a single genomic locus; although the sequenced repeats differ in up to 27 of 228 bases per repeat, they encode identical amino acid sequences. As this predicted amino acid sequence differs in only 3 of 76 residues from that of ubiquitin in higher eukaryotes, ubiquitin is apparently the most conserved of known proteins.     

Dependence of Drosophila wing imaginal disc cytonemes on Decapentaplegic

The anterior/posterior (A/P) and dorsal/ventral (D/V) compartment borders that subdivide the wing imaginal discs of Drosophila third instar larvae are each associated with a developmental organizer. Decapentaplegic (Dpp), a member of the transforming growth factor-beta (TGF-beta) superfamily, embodies the activity of the A/P organizer. It is produced at the A/P organizer and distributes in a gradient of decreasing concentration to regulate target genes, functioning non-autonomously to regulate growth and patterning of both the anterior and posterior compartments. Wingless (Wg) is produced at the D/V organizer and embodies its activity. The mechanisms that distribute Dpp and Wg are not known, but proposed mechanisms include extracellular diffusion, successive transfers between neighbouring cells, vesicle-mediated movement, and direct transfer via cytonemes. Cytonemes are actin-based filopodial extensions that have been found to orient towards the A/P organizer from outlying cells. Here we show that in the wing disc, cytonemes orient towards both the A/P and D/V organizers, and that their presence and orientation correlates with Dpp signalling. We also show that the Dpp receptor, Thickveins (Tkv), is present in punctae that move along cytonemes. These observations are consistent with a role for cytonemes in signal transduction.     

Segment-specific expression of a zinc-finger gene in the developing nervous system of the mouse

The process of segmentation, in which repeated homologous structures are generated along the anterior-posterior axis of the embryo is a widespread mechanism in animal development. In vertebrates, segmentation is most apparent in the somites and the peripheral nervous system, but the existence of repetitive bulges, termed neuromeres, in the early neural epithelium of vertebrates suggests that the CNS may also be segmented. Consistent with this, cranial ganglia and certain neurons are associated with specific hindbrain neuromeres. Here, we report that Krox-20, a zinc-finger gene, is expressed in two alternate neuromeres in the mouse early hindbrain. This pattern subsequently decays and Krox-20 is transiently expressed in specific hindbrain nuclei. In addition, Krox-20 is expressed in early neural crest cells, and then in the neural crest-derived boundary caps, glial components of the cranial and spinal ganglia. The demonstration that neuromeres are domains of gene expression provides molecular evidence for the segmentation of the CNS.     

Sequence of a Drosophila segmentation gene: protein structure homology with DNA-binding proteins

Mutations in the fushi tarazu (ftz) locus of Drosophila result in embryos with half the usual number of body segments. The sequences of the wild-type gene, a temperature-sensitive allele and a dominant mutant allele are presented. A portion of the conserved protein domain present in ftz and several homoeotic genes resembles the DNA-binding region of prokaryotic DNA-binding proteins, and is also similar to products of the yeast mating-type locus.     

Leukocytes express a novel gene encoding a putative transmembrane protein-kinase devoid of an extracellular domain

Tyrosine-specific phosphorylation of proteins is a key to the control of diverse pathways leading to cell growth and differentiation. The protein-tyrosine kinases described to date are either transmembrane proteins having an extracellular ligand binding domain or cytoplasmic proteins related to the v-src oncogene. Most of these proteins are expressed in a wide variety of cells and tissues; few are tissue-specific. Previous studies have suggested that lymphokines could mediate haematopoietic cell survival through their action on glucose transport, regulated in some cells through the protein-tyrosine kinase activity of the insulin receptor. We have investigated the possibility that insulin receptor-like genes are expressed specifically in haematopoietic cells. Using the insulin receptor-related avian sarcoma oncogene v-ros as a probe, we have isolated and characterized the complementary DNA of a novel gene, ltk (leukocyte tyrosine kinase). The ltk gene is expressed mainly in leukocytes, is related to several tyrosine kinase receptor genes of the insulin receptor family and has unique structural properties: it apparently encodes a transmembrane protein devoid of an extracellular domain. Two candidate ltk proteins have been identified with antibodies in the mouse thymus, and have properties indicating that they are integral membrane proteins. These features suggest that ltk could be a signal transduction subunit for one or several of the haematopoietic receptors.