A new moderately repetitive rat DNA sequence detected by a cloned 4.5 SI DNA

4.5 SI RNA is an abundant, noncapped, small nuclear RNA found in rodent cells. The 4.5 SI RNA is 98 or 99 nucleotides long and contains no modified nucleotides; it is synthesized by RNA polymerase III, is partly hydrogen-bonded to poly(A+) hnRNA, and was the first small nuclear RNA to be purified and sequenced (Busch, H., Reddy, R., Ruthblum, L., and Choi, Y. C. (1982) Annu. Rev. Biochem. 51, 617-654). In studies on the structure and organization of genes coding for this abundant RNA, it was found that this RNA is homologous to an apparently novel family of repetitive sequences. Two clones were characterized; one clone showed that its sequence is identical to the RNA in the first 92 residues and differed only in the last six nucleotides. In addition, the 3'-end of the sequence contained an A,T-rich region, and the sequence was flanked by a 15-nucleotide long direct repeat of AAAATATAGACACTG. The second clone characterized contained nucleotide sequences 1-57 corresponding to the RNA and was flanked by a 15-nucleotide long direct repeat. The structural features of these two DNAs are consistent with RNA-mediated DNA synthesis and integration of this DNA into the genome at random sites. It is estimated that there are about 10,000 copies of this family of sequences in the haploid rat genome.     

Molecular cloning and nucleotide sequence of cDNA encoding hydroxyindole O-methyltransferase of bovine pineal glands

Messenger RNA for hydroxyindole O-methyltransferase (EC 2.1.1.4) was partially purified from poly(A)+ RNA isolated from bovine pineal glands by sucrose density gradient centrifugation. The enriched mRNA was used to prepare a cDNA library by use of expression vector lambda gt11. The library was screened with monoclonal antibodies to the enzyme, and three cDNA clones were isolated. These cloned cDNAs cross-hybridized with one another, and their fusion proteins reacted to the monoclonal antibodies with different binding properties. Hydroxyindole O-methyltransferase enzymatic activity was demonstrated in the bacteria lysate infected with lambda HIOMT-A16, the clone that contained the longest insert. An almost full-length cDNA clone was isolated from lambda gt10 cDNA library by use of the lambda HIOMT-A16 cDNA as a probe. The primary structure of hydroxyindole O-methyltransferase was determined by analyzing the nucleotide sequence of the cDNAs. It consisted of 1939 nucleotides including a 1050-nucleotide region coding for 350 amino acids. RNA transfer blot analysis indicated that mRNA encoding hydroxyindole O-methyltransferase was present only in the pineal gland and not in the brain, retina, and liver of cow.     

Molecular cloning, nucleotide sequence, and tissue distribution of malonyl-CoA decarboxylase

Malonyl-CoA decarboxylase was purified from goose uropygial gland, reduced, carboxymethylated, and digested with trypsin. Several peptides were purified by high performance liquid chromatography and their amino acid sequences determined. Oligonucleotide probes were prepared based on their amino acid sequences. Size-selected RNA from the goose uropygial gland was used to construct cDNA libraries in lambda gt11 and pUC9 vectors. Immunological screening of the lambda gt11 cDNA library yielded one clone, lambda DC1, which contained a 2.2-kilobase pair insert; hybridization with the synthetic oligonucleotide probes confirmed its identity as malonyl decarboxylase. Screening of the pUC9 cDNA library with the insert of lambda DC1 as a probe detected one clone, pDC2, with an insert of 2.9 kilobase pairs. The nucleotide sequences of the two cDNAs revealed an open reading frame encoding a polypeptide of 462 amino acids. The deduced amino acid sequence was confirmed as malonyl-CoA decarboxylase by matching it to the amino acid sequences of three tryptic peptides derived from mature enzyme. Northern blot analysis of mRNA from goose brain, kidney, liver, lung, and gland revealed malonyl-decarboxylase mRNA of 3000 nucleotides. Since clone pDC2 contains a 2928-nucleotide insert, it represents nearly the full length of mRNA. Brain, kidney, lung, and liver contained less than 1% of the malonyl-CoA decarboxylase mRNA in the gland. Southern blot analysis of genomic DNA showed a single band in both liver and gland, suggesting that malonyl-CoA decarboxylase is a single copy gene.     

Morquio disease: isolation, characterization and expression of full-length cDNA for human N-acetylgalactosamine-6-sulfate sulfatase.

We cloned and sequenced a full-length cDNA of human placental N-acetylgalactosamine-6-sulfate sulfatase, the enzyme deficient in Morquio disease. The 2339-nucleotide sequence contained 1566 nucleotides which encoded a polypeptide of 522 amino acid residues. The deduced amino acid sequence was composed of a 26-amino acid N-terminal signal peptide and a mature polypeptide of 496 amino acid residues including two potential asparagine-linked glycosylation sites. Expression of the cDNA in transfected deficient fibroblasts resulted in higher production of this sulfatase activity than in untransfected deficient fibroblasts. The cDNA clone was hybridized to only a 2.3-kilobase species of RNA in human fibroblasts. The amino acid sequence of N-acetylgalactosamine-6-sulfate sulfatase showed a high degree of homology with those of other sulfatases such as human arylsulfatases A, B or C, glucosamine-6-sulfatase, iduronate-2-sulfatase and sea urchin arylsulfatase.     

Characterization and sequence analysis of a developmentally regulated putative cell wall protein gene isolated from soybean

A cDNA clone, pTU04, which hybridizes to two different sizes of mRNA on Northern blots was isolated from soybean suspension culture cell poly(A) RNA. Northern analysis reveals that meristematic tissue produces a 1050-nucleotide mRNA while quiescent mature cells produce primarily a 1220-nucleotide mRNA homologous to pTU04. The cDNA and its corresponding genomic clone have been partially characterized. The nucleotide sequence of the gene predicts a proline-rich protein, designated SbPRP1, which contains a signal peptide sequence and 43 repeats of a sequence consisting primarily of Pro-Pro-Val-Tyr-Lys (CCA-CCA-GTT-TAC-AAG). From nuclease S1 and hybrid-select translation analyses, the cDNA clone pTU04 appears to represent the mRNA for the mature tissue 1220-nucleotide RNA observed on Northern blots. Although there is no direct proof that the encoded protein is a cell wall protein, it has the properties similar to previously isolated cell wall proteins: 1) it is very basic with a high content of Pro, Tyr, and Lys; 2) it has similar hydropathic properties; and 3) its repeating unit shares sequence homology with that of more highly characterized cell wall proteins, generally termed extensin (Chen, J., and Varner, J. E. (1985) EMBO J. 4, 2145-2151; Smith, J. J., Muldoon, E. P., Willard, J. J., and Lamport, D. T. A. (1986) Phytochemistry 25, 1021-1030.     

Nucleotide sequence and expression of a Drosophila metallothionein

A Drosophila melanogaster cDNA clone was isolated based on its more intense hybridization to RNA sequences from copper-fed larvae than from control larval RNA. This clone showed strong hybridization to mouse metallothionein I cDNA at reduced stringency. Its nucleotide sequence includes an open reading segment which codes for a 40-amino acid protein; this protein is identified as metallothionein based on its similarity to the amino-terminal portion of mammalian and crab metalloproteins. The 10 cysteine residues present occur in five pairs of near vicinal cysteines (Cys-X-Cys). This cDNA sequence hybridized to a 400-nucleotide polyadenylated RNA whose presence in the cells of the alimentary canal of larvae was stimulated by ingestion of cadmium or copper; in other tissues this RNA was present at much lower levels. Mercury, silver, and zinc induced metallothionein to a lesser extent. The level of metallothionein RNA increased very soon after the initiation of metal treatment and reached a maximum after approximately 36 h.     

Characterization of a novel synGAP isoform, synGAP-beta

We cloned a cDNA encoding a novel synGAP, synGAP-d (GenBank(TM) accession number ), from a rat brain cDNA library. The clone consisted of 4801 nucleotides with a coding sequence of 3501 nucleotides, encoded a protein consisting of 1166 amino acids with >99% homology with 1092 amino acid overlaps to synGAP, and contained a 13-nucleotide insertion to the previously reported synGAP mRNAs, which suggested that the clone was a splice variant of synGAP. We also found that there are at least seven variants in the 3' portion of the synGAP mRNA and that they encoded five different protein isoforms. The coding sequence of these C-terminal variants were classified into alpha1, alpha2, beta1, beta2, beta3, beta4, and gamma, and synGAP-d was classified as the beta1 form. The previously reported synGAPs (synGAP-a, -b, and -c and p135synGAP) can be classified as the alpha1 isoform. All isoforms were expressed specifically in the brain. Unexpectedly, the beta isoform, which lacks a C-terminal PSD-95-binding motif ((S/T)XV), was more restricted to the postsynaptic density fraction than the motif-containing alpha1 isoform. The beta isoform did not interact with PSD-95 but specifically interacted with a nonphosphorylated alpha subunit of Ca(2+)/calmodulin-dependent protein kinase II through its unique C-terminal tail.     

A Sequence-Specific RNA-Binding Protein Complements Apobec-1 To Edit Apolipoprotein B mRNA

The editing of apolipoprotein B (apo-B) mRNA involves the site-specific deamination of cytidine to uracil. The specificity of editing is conferred by an 11-nucleotide mooring sequence located downstream from the editing site. Apobec-1, the catalytic subunit of the editing enzyme, requires additional proteins to edit apo-B mRNA in vitro, but the function of these additional factors, known as complementing activity, is not known. Using RNA affinity chromatography, we show that the complementing activity binds to a 280-nucleotide apo-B RNA in the absence of apobec-1. The activity did not bind to the antisense strand or to an RNA with three mutations in the mooring sequence. The eluate from the wild-type RNA column contained a 65-kDa protein that UV cross-linked to apo-B mRNA but not to the triple-mutant RNA. This protein was not detected in the eluates from the mutant or the antisense RNA columns. Introduction of the mooring sequence into luciferase RNA induced cross-linking of the 65-kDa protein. A 65-kDa protein that interacted with apobec-1 was also detected by far-Western analysis in the eluate from the wild-type RNA column but not from the mutant RNA column. For purification, proteins were precleared on the mutant RNA column prior to chromatography on the wild-type RNA column. Silver staining of the affinity-purified fraction detected a single prominent protein of 65 kDa. Our results suggest that the complementing activity may function as the RNA-binding subunit of the holoenzyme.     

cDNA克隆p14－6肿瘤抑制功能的分子机制

本文探讨了具有肿瘤抑制功能的ｃＤＮＡ克隆Ｐ１４－６（即人白细胞介素６核转录因子ＮＦ－ＩＬ６的３’非翻译区）的ＲＮＡ转录物与回复相关蛋白ＢＮＦ的相互作用,发现该ＲＮＡ与ＢＮＦ的相互作用位点为其３’侧的富Ｕ序列内的１个２４核苷酸片段;并发现ＢＮＦ系一群蛋白质,它们可能先相互结合成１个蛋白质复合物,然后再与ＲＮＡ位点作用．其中可能只有１个蛋白质（Ｒ６２）直接与该ＲＮＡ结合。