Polynucleotide sequence analysis by sequential base elimination: 3'-terminus of phage Q-beta RNA

The nucleotide sequence of a 3'-terminal fragment obtained by ribonuclease T(1) hydrolysis of the ribonucleic acid from bacteriophage Qbeta has been determined by an improved method of sequence analysis which involves sequential removal of bases by periodate oxidation and beta-elimination. The results obtained from ten such oxidation-elimination cycles and from the alkaline hydrolysis of the remaining oligonucleotide indicate that the first 16 nucleotides at the 3'-terminus of this ribonucleic acid have the sequence: -G-C-C-C-U-C-U-C-U-C-C-U-C-C-C-A.     

5S RNA synthesized by Escherichia coli in presence of chloramphenicol: different 5'-terminal sequences

Escherichia coli cells, grown in the presence of chloramphenicol, synthesize a low molecular weight RNA (CM-5S RNA) not bound to ribosomes which is similar to ribosomal 5S RNA. Oligonucleotide patterns derived from ribonuclease digests of 5S RNA and of CM-5S RNA are indistinguishable except that the 5'-terminal oligonucleotides differ. Whereas the nucleotide sequence of the 5'-terminus of normal 5S RNA is (p)U(p)G-, there are three alternate sequences of the 5'-terminus of CM-5S RNA: (p)U(p)U(p)G-, (p)U(p)U(p)U(p)G-, and (p)A(p)U(p)U(p)U(p)G-.     

Genetic variation in the human insulin gene

Four recombinant lambda phages containing nucleotide sequences complementary to a cloned human preproinsulin DNA probe have been isolated from human DNA. Restriction analyses in conjunction with Southern hybridizations reveal two types of gene sequences. One isolate of each type was subjected to complete nucleotide sequence determination. The sequences contain the entire preproinsulin messenger RNA region, two intervening sequence. 260 nucleotides upstream from the messenger RNA capping site, and 35 nucleotides beyond the polyadenylate attachment site. Our results strongly suggest that these two gene types are allelic variants of a single insulin gene.     

The context effect does not require a fourth base pair

The translational activity of a transfer RNA at a codon varies at different message sites, although the codon does not vary. The source of this effect, which may help to determine the level of gene expression, is generally agreed to be in nearby message sequences. By making every possible nucleotide combination between position 33 of the transfer RNA and the major context nucleotide of the message, it was shown that base-pairing between the two nucleotides is not the source of this context effect on translation in vivo.     

HTLV x-gene product: requirement for the env methionine initiation codon

The human T-cell leukemia viruses (HTLV) are replication-competent retroviruses whose genomes contain gag, pol, and env genes as well as a fourth gene, termed x, which is believed to be the transforming gene of HTLV. The product of the x gene is now shown to be encoded by a 2.1-kilobase messenger RNA derived by splicing of at least two introns. By means of S1 nuclease mapping of this RNA and nucleic acid sequence analysis of a complementary DNA clone, the complete primary structure of the x-gene product has been determined. It is encoded by sequences containing the env initiation codon and one nucleotide of the next codon spliced to the major open reading frame of the HTLV-I and HTLV-II x gene.     

Ribozyme-catalyzed transcription of an active ribozyme

A critical event in the origin of life is thought to have been the emergence of an RNA molecule capable of replicating a primordial RNA "genome." Here we describe the evolution and engineering of an RNA polymerase ribozyme capable of synthesizing RNAs of up to 95 nucleotides in length. To overcome its sequence dependence, we recombined traits evolved separately in different ribozyme lineages. This yielded a more general polymerase ribozyme that was able to synthesize a wider spectrum of RNA sequences, as we demonstrate by the accurate synthesis of an enzymatically active RNA, a hammerhead endonuclease ribozyme. This recapitulates a central aspect of an RNA-based genetic system: the RNA-catalyzed synthesis of an active ribozyme from an RNA template.     

The primary structure and heterogeneity of tau protein from mouse brain

Tau protein is a family of microtubule binding proteins, heterogeneous in molecular weight, that are induced during neurite outgrowth and are found prominently in neurofibrillary tangles in Alzheimer's disease. The predicted amino acid sequences of two forms of tau protein from mouse brain were determined from complementary DNA clones. These forms are identical in their amino-terminal sequences but differ in their carboxyl-terminal domains. Both proteins contain repeated sequences that may be tubulin binding sites. The sequence suggests that tau is an elongated molecule with no extensive alpha-helical or beta-sheet domains. These complementary DNAs should enable the study of various functional domains of tau and the study of tau expression in normal and pathological states.     

Nucleotide sequence of an RNA from cells infected with adenovirus 2

The principal nucleotide sequence of an RNA in KB cells infectedwith adenovirus 2 has been determined. As isolated, the molecule shows some terminal and internal heterogeneity. The sequence permits extensive base pairing and contains prominent repeating sequences. A portion of the sequence resembles a sequence found in several transfer RNA's.     

Polyadenylic acid sequences in the virion RNA of poliovirus and Eastern Equine Encephalitis virus

Poliovirus virion RNA contains a single covalently bound sequence of polyadenylic acid which is approximately 49 nucleotides long. A single, slightly longer polyadenylic acid sequence is contained in Eastern Equine Encephalitis virus RNA. Since these viruses are otherwise dissimilar these sequences may play a common role in viral replication, possibly in translation of the viral RNA.     

A ligase-mediated gene detection technique

An assay for the presence of given DNA sequences has been developed, based on the ability of two oligonucleotides to anneal immediately adjacent to each other on a complementary target DNA molecule. The two oligonucleotides are then joined covalently by the action of a DNA ligase, provided that the nucleotides at the junction are correctly base-paired. Thus single nucleotide substitutions can be distinguished. This strategy permits the rapid and standardized identification of single-copy gene sequences in genomic DNA.     

Nucleotide sequence of the "denaturable" leucine transfer RNA from yeast

The nucleotide sequence of " denaturable"leucine acceptor transfer RNA (tRNA(Leu)(3)) from baker's yeast was determined on (32)P-labeled material. The molecule is 85 nucleotides long and can be folded into the "cloverleaf" model for secondary structure. The basis on which the sequence was deduced from the products of complete enzymatic digestion, prior to its unambiguous determination, is presented.     

引物原位标记技术及其应用

本文综述了近几个来引物原位标记技术的发展和应用，并以检测猪染色体端位序列为例，介绍了这项技术的基本原理和实验程序。现已表明，引物原位标记技术不仅可以应用于检测染色体特定的ＤＮＡ序列，而且可以应用于在细胞和组织切片上检测特定的ＲＮＡ序列。     

Human endothelial cell growth factor: cloning, nucleotide sequence, and chromosome localization

Several of the endothelial cell polypeptide mitogens that have been described probably play a role in blood vessel homeostasis. Two overlapping complementary DNA clones encoding human endothelial cell growth factor (ECGF) were isolated from a human brain stem complementary DNA library. Southern blot analysis suggested that there is a single copy of the ECGF gene and that it maps to human chromosome 5 at bands 5q31.3 to 33.2 A 4.8-kilobase messenger RNA was present in human brain stem messenger RNA. The complete amino acid sequence of human ECGF was deduced from the nucleic acid sequence of these clones; it encompasses all the well-characterized acidic endothelial cell polypeptide mitogens described by several laboratories. The ECGF-encoding open reading frame is flanked by translation stop codons and provides no signal peptide or internal hydrophobic domain for the secretion of ECGF. This property is shared by human interleukin-1, which is approximately 30 percent homologous to ECGF.     

Neural cell adhesion molecule: structure, immunoglobulin-like domains, cell surface modulation, and alternative RNA splicing

The neural cell adhesion molecule, N-CAM, appears on early embryonic cells and is important in the formation of cell collectives and their boundaries at sites of morphogenesis. Later in development it is found on various differentiated tissues and is a major CAM mediating adhesion among neurons and between neurons and muscle. To provide a molecular basis for understanding N-CAM function, the complete amino acid sequences of the three major polypeptides of N-CAM and most of the noncoding sequences of their messenger RNA's were determined from the analysis of complementary DNA clones and were verified by amino acid sequences of selected CNBr fragments and proteolytic fragments. The extracellular region of each N-CAM polypeptide includes five contiguous segments that are homologous in sequence to each other and to members of the immunoglobulin superfamily, suggesting that interactions among immunoglobulin-like domains form the basis for N-CAM homophilic binding. Although different in their membrane-associated and cytoplasmic domains, the amino acid sequences of the three polypeptides appear to be identical throughout this extracellular region (682 amino acids) where the binding site is located. Variations in N-CAM activity thus do not occur by changes in the amino acid sequence that alter the specificity of binding. Instead, regulation is achieved by cell surface modulation events that alter N-CAM affinity, prevalence, mobility, and distribution on the surface. A major mechanism for modulation is alternative RNA splicing resulting in N-CAM's with different cytoplasmic domains that differentially interact with the cell membrane. Such regulatory mechanisms may link N-CAM binding function with other primary cellular processes during the embryonic development of pattern.     

RNA-catalyzed RNA polymerization: accurate and general RNA-templated primer extension

The RNA world hypothesis regarding the early evolution of life relies on the premise that some RNA sequences can catalyze RNA replication. In support of this conjecture, we describe here an RNA molecule that catalyzes the type of polymerization needed for RNA replication. The ribozyme uses nucleoside triphosphates and the coding information of an RNA template to extend an RNA primer by the successive addition of up to 14 nucleotides-more than a complete turn of an RNA helix. Its polymerization activity is general in terms of the sequence and the length of the primer and template RNAs, provided that the 3' terminus of the primer pairs with the template. Its polymerization is also quite accurate: when primers extended by 11 nucleotides were cloned and sequenced, 1088 of 1100 sequenced nucleotides matched the template.     

大麦条纹花叶病毒中国株基因组RNA1的全长序列分析

 【目的】获得大麦条纹花叶病毒中国株（BSMV-CH）基因组RNA1的全长cDNA,进行序列分析,明确与国外报道株系间的亲缘关系和分类地位。【方法】以自然侵染BSMV-CH的大麦叶片中提取的总RNA为模板,用RT-PCR方法获得BSMV-CH RNA1的全长cDNA,克隆到载体pMD18并测序,用DNAstar软件将获得的序列与GenBank登录的BSMV RNA1序列进行对比,分析各株系序列RNA1之间的同源性,构建系统发育树分析BSMV-CH与国外株系间的亲缘关系。【结果】BSMV-CH的RNA1全长为3 795 bp（GenBank注册号：AY789693）,基因组RNA1 5′端有一个甲基化的帽子结构,3′端有一个PolyA结构和一段239 bp长的类似tRNA的保守序列,BSMV-CH基因组RNA1含有一个长度为3 417 bp的ORF,推测可编码一个1 138个氨基酸的复制酶基因。BSMV-CH的基因组RNA1与国外报道其它株系核苷酸的同源性为95.4%～95.6%。复制酶基因同其它株系核苷酸和氨基酸的同源性分别为94.9%～95.2%和96.5%～96.8%,PolyA的长度比其它株系明显要长。【结论】BSMV-CH和CV42与CV17、ND18、TSE及TSG间的亲缘关系明显低于CV17、ND18、TSG和TSE间的亲缘关系;BSMV-CH与CV42表现相对较近的亲缘关系,但BSMB-CH是不同于CV42的一个BSMV新株系。     

RNA as an RNA polymerase: net elongation of an RNA primer catalyzed by the Tetrahymena ribozyme

A catalytic RNA (ribozyme) derived from an intervening sequence (IVS) RNA of Tetrahymena thermophila will catalyze an RNA polymerization reaction in which pentacytidylic acid (C5) is extended by the successive addition of mononucleotides derived from a guanylyl-(3',5')-nucleotide (GpN). Cytidines or uridines are added to C5 to generate chain lengths of 10 to 11 nucleotides, with longer products being generated at greatly reduced efficiency. The reaction is analogous to that catalyzed by a replicase with C5 acting as the primer, GpNs as the nucleoside triphosphates, and a sequence in the ribozyme providing a template. The demonstration that an RNA enzyme can catalyze net elongation of an RNA primer supports theories of prebiotic RNA self-replication.     

Human insulin-degrading enzyme shares structural and functional homologies with E. coli protease III

A proteinase with high affinity for insulin has been proposed to play a role in the cellular processing of this hormone. A complementary DNA (cDNA) coding for this enzyme has been isolated and sequenced. The deduced amino acid sequence of the enzyme contained the sequences of 13 peptides derived from the isolated protein. The cDNA could be transcribed in vitro to yield a synthetic RNA that in cell-free translations produced a protein that coelectrophoresed with the native proteinase and could be immunoprecipitated with monoclonal antibodies to this enzyme. The deduced sequence of this proteinase did not contain the consensus sequences for any of the known classes of proteinases (that is, metallo, cysteine, aspartic, or serine), but it did show homology to an Escherichia coli proteinase (called protease III), which also cleaves insulin and is present in the periplasmic space. Thus, these two proteins may be members of a family of proteases that are involved in intercellular peptide signaling.     

A labile phosphodiester bond at the ligation junction in a circular intervening sequence RNA

The excised intervening sequence of the Tetrahymena ribosomal RNA precursor mediates its own covalent cyclization in the absence of any protein. The circular molecule undergoes slow reopening at a single phosphodiester bond, the one that was formed during cyclization. The resulting linear molecule has 5'-phosphate and 3'-hydroxyl termini; these are unusual products for RNA hydrolysis but are typical of the other reactions mediated by this molecule. The reopened circle retains cleavage-ligation activity, as evidenced by its ability to undergo another round of cyclization and reopening. The finding that an RNA molecule can be folded so that a specific phosphate can be strained or activated helps to explain how the activation energy is lowered for RNA self-splicing. The proposed mechanisms may be relevant to several other RNA cleavage reactions that are RNA-mediated.