Location of the genes coding for 18S and 28S ribosomal RNA in the human genome

³H-rRNA obtained from Xenopus laevis tissue cultured cells, or a ³H-cRNA made from Xenopus ribosomal DNA, was used for heterologous in situ hybridisation with human lymphocyte metaphase chromosomes. Prior to hybridisation, chromosome spreads were stained with Quinacrine and selected cells showing good Q-banding photographed; the same cells were then rephotographed after autoradiography and pairs of photographs for each cell were used to make dual karyotypes. The chromosomes within each karyotype were divided into equal sized segments (approx. 0.7 μ), with a fixed number of segments for each chromosome type. The distribution of silver grains between segments showed that the ³H-RNAs hybridised specifically to the nucleolar organising regions of the D and G group chromosomes with no other sites of localised labelling in the complement. Control experiments showed no localisation, with insignificant labelling, when metaphase spreads were incubated in a mixture containing Xenopus ³H-rRNA and competing cold human (HeLa) rRNA. Filter hybridisation experiments on isolated human DNA showed that the Xenopus derived ³H-RNAs hybridised to a fraction of human DNA which was on the heavy side of the main DNA peak and that these RNAs were competed out in the presence of excess cold human rRNA, confirming the specificity of the heterologous hybridisation. In situ hybridisation experiments were also carried out on cells from individuals with one chromosome pair showing heteromorphism for either a very long stalk (nucleolar constriction) subtending a satellite, or a large satellite. It was shown that the chromosome with the large stalk hybridised four times as much ³H-rRNA as its homologue, whereas differences in the sizes of the subtended satellites did not materially affect hybridisation levels indicating that rDNA is located in the stalks and not the satellites. The amount of ³H-rRNA hybridised differs between chromosomes and individuals; these differences are heritable and rDNA can be detected by in situ hybridisation in all three chromosomes number 21 in cells from Down's patients and in translocated chromosomes conta.ining a nucleolar constriction. Different D and G group chromosomes which hybridised equal amounts of ³H-rRNA participated in rosette associations at metaphase in a random fashion in some individuals and in a non-random fashion in others. In all individuals studied chromosomes with large amounts of rDNA were not found to be preferentially involved in association. It was therefore concluded that the probability of a chromosome being involved in the formation of a common nucleolus is not a simple function of its rDNA content and other possible factors are considered.     

Location of genes coding for 18S and 28S ribosomal RNA within the genome of Mus musculus

Cytological detection of cistrons coding for 18S and 28S ribosomal RNA (rRNA) within the genome of Mus musculus inbred strain SEC/1ReJ was accomplished using the technique of in situ hybridization. Metaphase chromosome spreads prepared from cultured fetal mouse cells were stained with quinacrine-HCl and photographed. After destaining, they were hybridized to Xenopus laevis tritiated 18S and 28S rRNA, specific activity 7.5 X 10(6) dpm/mug. Silver grains clustered over specific chromosomes were readily apparent after 4 months of autoradiographic exposure. The identity of the labelled chromosomes was established by comparing the autoradiographs to quinacrine photographs showing characteristic fluorescent banding of the chromosomes in each metaphase spread. The 18S and 28S rRNA was found to hybridize to chromosomes 12, 18, and 16. Statistical analysis of the grain distribution over 26 spreads revealed that the three chromosomes were significantly labelled. Grains over these chromosomes were concentrated in an area immediately distal to the centromere, a region which in chromosomes 12 and 18 in this particular strain is the site of a secondary constriction. The relative size of the secondary constrictions, long and thus prominent on chromosome 12, obvious but shorter on 18, and indistinguishable on chromosome 16, correlated with the average number of grains observed over the centromeric region of these chromosomes, 2.5, 1.0, and 0.78, respectively.     

Hybridization maps of early and late messenger RNA sequences on the adenovirus type 2 genome.

Specific fragments of adenovirus type 2 DNA, generated by cleavage with restriction endonucleases endoR.EcoRI, endoR.HpaI and endoR.HindIII were used in hybridization-mapping experiments. The complementary strands of individual cleavage fragments were separated by the method of Tibbetts &; Pettersson (1974). Liquid hybridizations were performed with ³²P-labeled separated strands of cleavage fragments and messenger RNA extracted from cells early and late after adenovirus infection. The fraction of each fragment strand which was represented in “early” and “late” messenger RNA was determined by chromatography on hydroxylapatite. Early messenger RNA was found to be derived from four widely separated regions, two on the 1- and two on the h-strand (h- and l- refer to the strand with heavy and light buoyant density in CsCl when complexed with poly(U, G)). Messenger RNA, present exclusively late after infection, is derived from several locations, predominantly from the l-strand with a major block of continuous sequences extending between positions 0.25 and 0.65 on the unit map of the adenovirus type 2 genome.     

DNA sequences from the adenovirus 2 genome

The sequence of 5,839 nucleotides from the adenovirus 2 genome has been determined and includes the regions between coordinates 32-44% and 66-71%. These regions contain the coding sequences for the 52,55K polypeptide, polypeptide IIIa, penton base, and the N terminus of the 100K polypeptide. Several additional unidentified open reading frames are present, including examples which overlap identified reading frames on the complementary strand and on the same strand. In conjunction with previously published sequences and those described in the accompanying papers (Akusj?rvi, G., Alestr?m, P., Pettersson, M., Lager, M., J?urnvall, H., and Pettersson, U. (1984) J. Biol. Chem. 259, 13976-13979; Alestr?m, P., Akusj?rvi, G., Lager, M., Yeh-kai, L., and Pettersson, U. (1984) J. Biol. Chem. 259, 13980-13985) a complete sequence of 35,937 nucleotide pairs can now be reconstructed for the adenovirus 2 genome.     

真核生物基因组中的非编码序列

真核生物基因组绝大部分是非编码序列;绝大部分非编码序列以高度重复序列的形式存在,如卫星、小卫星、微卫星、长散布元件、短散布元件等;内含子、３’不译区作为结构基因的一部分被一同转录;ＲＮＡ基因转录具有明确功能的ＲＮＡ分子;顺式作用元件是目前已知的具有重要调控功能的非编码序列;非编码序列的存在与真核生物基因表达调控密切相关;目前非编码序列的研究已引起广泛的科学关注,利用数理方法研究其遗传信息的储存方式     

Comparison of the sequences and coding of La Crosse and snowshoe hare bunyavirus S RNA species.

The sequence of the S RNA of La Crosse bunyavirus was deduced from analyses of DNA copies cloned in the Escherichia coli plasmid pBR322. The S RNA is 984 nucleotides in length, has a base ratio of 31.8% U, 27.0% A, 23.2% C, and 18.0% G, and codes for two distinct gene products that are read from overlapping reading frames in the viral complementary strand. The larger gene product (N, 26.5 x 10(3) daltons) contains 235 amino acids, and the smaller gene product (NSS, 10.4 x 10(3) daltons) has 92 amino acids. Comparisons with the published sequences of the related snowshoe hare bunyavirus S RNA and its gene products (Bishop et al., Nucleic Acids Res. 10:3703-3713, 1982) indicate that there are a total of 114 nucleotide differences (6 additions or deletions and 108 substitutions). Also, there are 22 amino acid differences between the N proteins and 12 amino acid differences between the NSS proteins of the two S RNAs.     

Location of the sequences coding for capsid proteins VP1 and VP2 on polyoma virus DNA.

The 19S and 16S polyoma virus late mRNAs have been separated on sucrose-formamide density gradients and translated in vitro. The 16S RNA codes only for polyoma capsid protein VP1, while the 19S RNA codes in addition for capsid protein VP2. Since the 19S and 16S species have been previously mapped on the viral genome, these results allow us to deduce the location of the sequences coding for VP1 and VP2. Comparison of the chain lengths of the capsid proteins with the size of the viral mRNAs coding for them suggests that VP1 and VP2 are entirely virus-coded. Purified polyoma 19S RNA directs the synthesis of very little VP1 in vitro, although it contains all the sequences required to code for the protein. The initiation site for VP1 synthesis which is located at an internal position on the messenger is probably inactive either because it is inaccessible or because it lacks an adjacent "capped" 5' terminus. Similar inactive internal initiation sites have been reported for other eucarotic viral mRNAs (for example, Semliki forest virus, Brome mosaic virus, and tobacco mosaic virus), suggesting that while eucaryotic mRNAs may have more than one initiation site for protein synthesis, only those sites nearer the 5' terminus of the mRNA are active.     

Organization of sequences related to U6 RNA in the human genome.

Small nuclear RNAs were isolated from human placenta and fractionated into individual molecular species. They were then iodinated with 125I and used as probes to screen the human genome. Of 2 x 10(4) recombinant phage clones screened, 22 clones hybridized with U6 RNA, suggesting that there were about 200 copies of this sequence family per haploid genome. Southern blots of these cloned DNAs digested with several restriction enzymes gave the following results: 1, each clone had only one fragment that carried the U6 sequence, 2, the lengths of these fragments varied from clone to clone. These observations indicate that U6 sequences exist as dispersed middle repetitive DNA, and that the sequences surrounding these loci vary. Two of the loci and their flanking regions were subcloned into plasmid and sequenced. Both of the loci showed microheterogeneity of mainly A/G and T/C, but had closely related sequences to U6 RNAs of rat or mouse. The divergence of the flanking regions begins immediately outside the loci. The implication on the microheterogeneity of the U6-related sequences is discussed.     

Influenza B virus genome: sequences and structural organization of RNA segment 8 and the mRNAs coding for the NS1 and NS2 proteins.

Double-stranded DNA derived from influenza B virus genome RNA segment 8, which codes for the NS1 and NS2 proteins, was constructed by hybridization of full-length cDNA copies of RNA segment 8 and of the NS1 mRNA. This DNA was cloned in plasmid pBR322 and sequenced. The NS1 mRNA (approximately 1,080 viral nucleotides) contains nonviral nucleotides at its 5' end and is capable of coding for a protein of 281 amino acids. Sequencing of the NS2 mRNA has shown that it contains an interrupted sequence of 655 nucleotides and is most likely synthesized by a splicing mechanism. The first approximately 75 virus-specific nucleotides at the 5' end of the NS2 mRNA are the same as are found at the 5' -end of the NS1 mRNA. This region contains the initiation codon for protein synthesis and coding information for 10 amino acids common to the two proteins. The approximately 350-nucleotide body region of the NS2 mRNA can be translated in the +1 reading frame, and the sequence indicates that the NS1 and NS2 protein-coding regions overlap by 52 amino acids translated from different reading frames. Thus, between the influenza A and B viruses, the organization of the NS1 and NS2 mRNAs and the sizes of the NS2 mRNA and protein are conserved despite the larger size of the influenza B virus RNA segment, NS1 mRNA, and NS1 protein.     

Yeast mitochondrial transfer RNA. Structure, coding properties and genome organization

The up-to-date data on mitochondrial tRNAs of yeast, their structures and peculiarities of these structures, anomalies of the mitochondrial genetic code and anticodons of tRNAs, the structure and number of tRNA genes are reviewed in the present paper. New information concerning 17 types of yeast mitochondrial tRNAs, deciphered by the authors of the paper are given; among them 8 types are first published. The likeness and differences of yeast mitochondrial tRNAs from their cytoplasmic counterparts are discussed by comparison with other organisms.     

Unique arrangement of coding sequences for 5 S, 5.8 S, 18 S and 25 S ribosomal RNA in Saccharomyces cerevisiae as determined by R-loop and hybridization analysis.

The arrangement of the coding sequences for the 5 S, 5.8 S, 18 S and 25 S ribosomal RNA from Saccharomyces cerevisiae was analyzed in λ-yeast hybrids containing repeating units of the ribosomal DNA. After mapping of restriction sites, the positions of the coding sequences were determined by hybridization of purified rRNAs to restriction fragments, by R-loop analysis in the electron microscope, and by electrophoresis of S₁ nuclease-treated rRNA/rDNA hybrids in alkaline agarose gels. The R-loop method was improved with respect to the length calibration of RNA/DNA duplexes and to the spreading conditions resulting in fully extended 18 S and 25 S rRNA R-loops. The qualitative results are: (1) the 5 S rRNA genes, unlike those in higher eukaryotes, alternate with the genes of the precursor for the 5.8 S, 18 S and 25 S rRNA; (2) the coding sequence for 5.8 S rRNA maps, as in higher eukaryotes, between the 18 S and 25 S rRNA coding sequences. The quantitative results are: (1) the tandemly repeating rDNA units have a constant length of 9060 ± 100 nucleotide pairs with one SstI, two HindIII and, dependent on the strain, six or seven EcoRI sites; (2) the 18 S and 25 S rRNA coding regions consist of 1710 ± 80 and 3360 ± 80 nucleotide pairs, respectively; (3) an 18 S rRNA coding region is separated by a 780 ± 70 nucleotide pairs transcribed spacer from a 25 S rRNA coding region. This is then followed by a 3210 ± 100 nucleotide pairs mainly non-transcribed spacer which contains a 5 S rRNA gene.