The 26S rRNA binding ribosomal protein equivalent to bacterial protein L11 is encoded by unspliced duplicated genes in Saccharomyces cerevisiae.

Transformant phages expressing L15, a yeast ribosomal protein which binds to 26S rRNA and interacts with the acidic ribosomal proteins, were isolated by screening a yeast cDNA expression library in lambda gt11 with specific monoclonal antibodies. Using yeast DNA HindIII fragments that hybridize with the cDNA insert from the L15-expressing clones, minilibraries were prepared in pUC18, which were afterward screened with the same cDNA probe. In this way, plasmids carrying two different types of genomic DNA inserts were obtained. The inserts were subcloned and sequenced and we found a similar coding sequence in both cases flanked by 5' and 3' regions with very low homology. Sequences homologous to the consensus TUF-binding UAS boxes are present in the 5' flanking regions of both genes. Southern analysis revealed the presence of two copies of the L15 gene in the Saccharomyces cerevisiae genome, which are located in different chromosomes. The encoded amino acid sequence corresponds, as expected, to protein L15 and shows a high similarity to bacterial ribosomal protein L11.     

Alternatively spliced transcripts of the Drosophila tramtrack gene encode zinc finger proteins with distinct DNA binding specificities.

A protein present in nuclear extracts of Drosophila embryos binds multiple sites in the promoter and genetically defined autoregulatory element of the pair-rule gene even-skipped (eve). We reported here the isolation of a cDNA encoding this binding activity, the sequence of which identifies it as the 69 kDa zinc finger tramtrack (ttk) protein. As ttk was previously implicated in controlling the expression of another pair-rule gene, fushi tarazu (ftz), our findings suggest that ttk plays a role in the regulation of at least two developmentally important genes. An additional ttk-related cDNA clone was isolated which gives rise to an 88 kDa protein with an alternative set of zinc fingers having a DNA binding specificity distinct from that of the 69 kDa protein. Both proteins were shown to be encoded by the ttk gene through alternative splicing, providing the first example of the use of this mechanism to generate related proteins with distinct DNA binding specificities. Whole mount in situ hybridization analysis revealed different patterns of embryonic expression of the two ttk mRNA isoforms.     

一种拟南芥ZIP蛋白全长cDNA的克隆及表达

利用异质DNA探针 ,从拟南芥叶片cDNA文库中筛选到一个阳性cDNA克隆———AT10 3。DNA序列分析表明 ,其编码产物含有一个亮氨酸拉链结构域和一个核定位信号肽。GenBank数据库搜寻结果显示 ,AT10 3未与任何已知功能基因有同源性 ,但与裂叶牵牛PNZIP、集胞藻的一个ORF、紫菜的一个ORF ,构成了一个在进化上非常保守的含有亮氨酸拉链结构域的新家族 ,极有可能是一新的核基因转录因子。AT10 3是一个单拷贝基因 ,在叶片组织中强烈表达并受光调节     

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.     

Amino acid sequence homologies between rabbit, rat, and human serum retinol-binding proteins

The main transporting protein for vitamin A in rabbit serum, the retinol-binding protein (RBP), was isolated and its amino acid sequence determined. Rabbit RBP was found to be highly homologous to human RBP, whose amino acid sequence was elucidated earlier, and to rat RBP. The rat RBP sequence was obtained by combining information deduced from the nucleotide sequences of two overlapping cDNA clones with the NH2-terminal sequence of the isolated protein determined by automated Edman degradation. The identity between the three proteins is approximately 90%. The high degree of homology between RBP molecules from different species is probably explained by the fact that RBP participates in at least three types of molecular interactions: in the binding of prealbumin, in the interaction with retinol, and in the recognition of a specific cell surface receptor. All these interactions should lead to a conservation of RBP structure. The amino acid differences between rabbit, rat, and human RBP are discussed in light of the recent elucidation of the three-dimensional structure of human RBP. Hybridization of a probe isolated from a rat RBP cDNA clone to restriction enzyme-digested genomic DNA from rat and mouse suggests that RBP is encoded by a single gene.     

Characterization and hetereologous expression of cDNAs encoding two novel closely related Ca(2+)-binding proteins in Dictyostelium discoideum

During a yeast two hybrid screen of a Dictyostelium cDNA library using the Ca(2+)-binding protein CBP1 as bait, we isolated a full-length cDNA encoding a novel Ca(2+)-binding protein (termed CBP4a). The protein is composed of 162 amino acids and contains four consensus EF-hands. PCR amplification of Dictyostelium genomic DNA using primers specific for the cDNA sequence resulted in the isolation of a gene encoding a different Ca(2+)-binding protein of 162 amino acids (designated CBP4b) with 90% amino acid sequence identity to CBP4a. Southern blot analysis confirmed the presence of two closely related genes in the Dictyostelium genome. CBP4a and CBP4b mRNAs are expressed at the same stages of development as CBP1 mRNA. In addition, both novel proteins bind (45)Ca(2+) and interact with CBP1 in vitro in a Ca(2+)-dependent manner.     

Heterogeneity of amelogenin mRNA in the bovine tooth germ

The amelogenins are a complex mixture of hydrophobic proteins that are the major organic component of developing enamel. To study the molecular mechanisms underlying the heterogeneity of the amelogenins we isolated cDNA clones encoding these proteins. The clones were definitively identified by hybrid-selected translation experiments and by comparison of the DNA sequence with the protein-derived amino acid sequence. Southern hybridization of bovine genomic DNA indicated that amelogenin is a single copy gene. However, Northern hybridization experiments distinctly showed two major species of mRNA, each of which were sufficiently large enough to encode the highest known molecular weight species of amelogenin proteins. Furthermore, immunoprecipitation of hybrid-selected translation products using isolated amelogenin cDNA showed multiple, translated protein products. These data are supportive of a differential mRNA processing mechanism involved in generating a heterogeneous family of amelogenin matrix proteins from a single gene.     

Characterization of a triplex DNA-binding protein encoded by an alternative reading frame of loricrin.

In an attempt to identify genes encoding triple-helical DNA-binding proteins, we performed South-Western screening of a human keratinocyte cDNA expression library using a purine (Pu)-rich triplex DNA probe. We isolated two independent clones containing part of the loricrin gene. Both were translated with a different reading frame than that of the loricrin protein, the major component of the cell envelope during normal keratinocyte cornification. The affinity of the encoded polypeptide for Pu-triplex DNA was confirmed by electrophoretic mobility shift assays using a bacterially expressed N-terminal loricrin deletion fused with the maltose-binding protein (MBP-LOR3ARF). Interactions between Pu-triplex DNA and MBP-LOR3ARF are characterized by a distribution of four increasingly slower mobility complexes, suggesting that multiple MBP-LOR3ARF molecules can recognize a single triplex. Binding was also observed between MBP-LOR3ARF and a pyrimidine-motif triplex DNA, although at lower affinity than Pu-triplex DNA. No apparent binding was observed between MBP-LOR3ARF and double-stranded DNA, suggesting that MBP-LOR3ARF is a bona fide Pu-triplex binding protein. Finally, purified specific rabbit antibodies against LORARF detected four human proteins with apparent molecular masses of 210, 110, 68, and 66 kDa on Western blot analysis. The 210-, 110-, and 68-kDa proteins also showed specific Pu-triplex DNA binding in a South-Western experiment, suggesting that LORARF shares common domains with other human Pu-triplex DNA-binding proteins.     

Nucleotide sequences of cloned cDNA fragments specific for six Xenopus laevis ribosomal proteins

We have previously constructed and selected six recombinant plasmids containing cDNA sequences specific for different ribosomal proteins of Xenopus laevis (Bozzoni et al., 1981). DNA cloned in these plasmids have been isolated and sequenced. Amino acid sequences of the corresponding portions of the proteins have been derived from DNA sequences; they are arginine- and lysine-rich as expected for ribosomal proteins. One of the cDNA sequences has an open reading frame also on the strand complementary to the one coding for the ribosomal protein; this fragment has inverted repeats twenty nucleotides long at the two ends. The codon usage for the six sequences appears to be non-random with some differences among the ribosomal proteins analysed.     

玉米21kD富硫种子贮存蛋白的cDNA克隆及其序列分析

利用逆转录－聚合酶链式反应（ＲＴ－ＰＣＲ）方法从玉米掖单－２０开花后１０ｄ的叶片中分离到２１ｋＤ玉米种子贮存蛋白ｃＤＮＡ（Ｎ２１ＫＺＹ）,并进行了序列分析．其编码蛋白包含２１１个氨基酸,极其富含甲硫氨酸,高达２７％;其Ｎ端有一个２１个氨基酸的信号肽．Ｎ２１ＫＺＹ及其编码蛋白和Ｃｈｕｉ等人分离的该基因的基因组克隆及其编码蛋白的同源性分别为９５．１％和９０．５％;两者的编码蛋白与玉米１０ｋＤ醇溶蛋白极其相似,其中间多出一个５４氨基酸的肽段和一个６氨基酸的肽段,这表明它们可能是来源于同一个祖先基因,后来通过基因重排、缺失或不均等交换等过程而形成的不同的蛋白质．     

Molecular characterisation and expression of a wound-inducible cDNA encoding a novel cinnamyl-alcohol dehydrogenase enzyme in lucerne (Medicago sativa L.)

A lucerne (alfalfa, Medicago sativa) stem cDNA library was screened with a cinnamyl-alcohol dehydrogenase (CAD) cDNA probe from tobacco (Nicotiana tabacum cv. Samsun). Two distinctly different cDNA clones (54% identical) were isolated and identified as putative CAD-encoding cDNAs by comparison of their nucleotide sequences with those of CAD-encoding DNA sequences from other plant species. One of the cDNAs, MsaCad2, was found to be 99.4% identical at the nucleotide level to the previously isolated lucerne cad cDNA which encodes a CAD isoform involved in lignin biosynthesis. The other cDNA, MsaCad1, has not been reported previously in lucerne, and encodes a protein related to the ELI3 class of elicitor-inducible defence-related plant proteins. The MsaCad1- and MsaCad2-encoded proteins were expressed in Escherichia coli and CAD1 was shown to be active with a range of cinnamyl, benzyl and aliphatic aldehyde substrates, while CAD2 was specific for the cinnamyl aldehydes only. Each of the respective genes is present as one or two copies. The MsaCad1 gene is expressed most actively in stem and floral tissue, whereas MsaCad2 is most actively expressed in stem, hypocotyl and root tissue. In stem tissue, expression of both genes occurs predominantly in internodes 4 and 5 (from the apex). MsaCad2, in contrast to MsaCad1, is not significantly expressed in the top three internodes of the stem. Both MsaCad1 and MsaCad2 are wound-inducible, and the wound-responsiveness of each gene is modulated by salicylic acid.     

Isolation of a novel ras gene from Trichomonas vaginalis: a possible evolutionary ancestor of the Ras and Rap genes of higher eukaryotes.

The Ras subfamily proteins are small, monomeric GTP-binding proteins with vital roles in regulating eukaryotic signal transduction pathways. Gene duplication and divergence have been postulated as the mechanism by which such family members have evolved their specific functions. A cDNA clone of TvRsp was isolated and sequenced from a cDNA expression library of the primitive eukaryote Trichomonas vaginalis. The genomic DNA corresponding to the cDNA sequence was amplified by PCR and sequenced. Sequence analysis suggested that TvRsp was an intronless gene. This gene encoded a protein of 181 amino acids and contained the 5 conserved G domains that designated it as a Ras or Rap subfamily member. However, the deduced amino acid sequence shared only 34%-37% overall identity with other Ras subfamily members of different species, and the presence of motifs characteristic of both the Ras and Rap families of GTPase confused the familial classification of this gene. Phylogenetic analysis showed its origins at the divergence point of the Ras/Rap families and suggested that TvRsp was a possible evolutionary ancestral gene of the ras/rap genes of higher eukaryotes. This information was of importance not only from the perspective of understanding the evolution and diversity of eukaryotic signal transduction pathways but also in providing a framework by which to understand protein processing in the growth and differentiation of single-celled microorganisms.     

Molecular characterization of a gene encoding a cysteine-rich protein preferentially expressed in anthers of Lycopersicon esculentum

The tomato (Lycopersicon esculentum Mill.) cDNA clone TomA5B was isolated by differential screening of a cDNA library prepared from anthers at late meiosis to tetrad formation. The 5B gene is present in a single copy in the tomato genome. Expression is developmentally regulated and tissue specific. RNA accumulation was detected from premeiosis through tetrad release in the tapetal cell layer of the anther with low levels of RNA detected in petals and early stages of pistil development. The protein deduced from the DNA sequence analysis is predicted to have a molecular mass of 11.1 kDa and a secretory signal sequence, suggesting it is a secreted protein. The deduced 5B protein has a pattern of cysteine residues that is similar to other proteins that have stamen-specific expression and to a superfamily of seed proteins. The 5B protein is unique in that there is no amino acid sequence similarity to other proteins beyond the similar cysteine motif.     

The chromosomal gene structure and two mRNAs for human granulocyte colony-stimulating factor.

Two different cDNAs for human granulocyte colony-stimulating factor (G-CSF) were isolated from a cDNA library constructed with mRNA prepared from human squamous carcinoma cells, which produce G-CSF constitutively. The nucleotide sequence analysis of both cDNAs indicated that two polypeptides coded by these cDNAs are different at one position where three amino acids are deleted/inserted. When the two cDNAs were introduced into monkey COS cells under the SV40 early promoter, both of them produced proteins having authentic G-CSF activity and some difference in the specific activity was suggested. A human gene library was then screened with the G-CSF cDNA and the DNA fragment containing the G-CSF chromosomal gene was characterized by the nucleotide sequence analysis. The human G-CSF gene is interrupted by four introns and a comparison of the structures of the two G-CSF cDNAs with that of the chromosomal gene indicated that the two mRNAs are generated by alternative use of two 5' splice donor sequences in the second intron of the G-CSF gene. When the G-CSF chromosomal gene was expressed in monkey COS cells by using the SV40 enhancer two mRNAs were detected by S1 mapping analysis.