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PubMed1991

Transcripts of simian virus 41 (SV41) matrix gene are exclusively dicistronic with the fusion gene which is also transcribed as a monocistron.

The complete nucleotide sequences of the matrix (M) and fusion (F) genes of simian virus 41 (SV41) were determined. Deduced amino acid sequences confirmed the close relationship of SV41 with human parainfluenza type 2 virus (PIV2). Analyses of noncoding regions between the F and the hemagglutinin-neuraminidase (HN) genes suggested the absence of the small hydrophobic gene, which is present between the F and the HN genes of simian virus 5 and mumps virus. It was striking that there was no apparent consensus gene end sequence between the M and the F genes and that the M gene was transcribed exclusively as a dicistron with the F gene. The number of monocistronic transcripts of the F gene was approximately half that of the dicistronic transcripts. However, the F protein of SV41 seemed to be efficiently translated, since viral multiplication and fusion from within were as efficient as in PIV2. These results suggest that the lack of a consensus gene end sequence resulted in the readthrough of viral RNA polymerases between the M and the F genes and that the initiation of F gene transcription could occur by newly entered polymerases independently of the polymerases that started the upstream M gene transcription.

Amino Acid Sequence↗

PubMed1991

Host-derived 5' ends and overlapping complementary 3' ends of the two mRNAs transcribed from the ambisense S segment of Uukuniemi virus.

Two mRNAs, coding for the N and NSS proteins, are transcribed from the small (S) Uukuniemi virus RNA segment by an ambisense strategy (J. F. Simons, U. Hellman, and R. F. Pettersson, J. Virol. 64:247-255, 1990). In this report, we describe the analysis of the 5' and 3' ends of the two mRNAs. Primer extension as well as cloning and sequencing of individual mRNAs showed that the 5' ends of both mRNAs contained nonviral sequences ranging from 7 to 25 residues in length (mean, 12 residues), indicating a cap-snatching mechanism similar to the one originally described for priming of influenza virus mRNA synthesis. In 35% of the cases, the first virion-specified nucleotide (an A residue) was substituted with a G residue. Between the translation termination codons of N and NSS, there is a 74-residue-long noncoding intergenic region (Simons et al., J. Virol. 64:247-255, 1990). Nuclease protection assays using both RNA and DNA hybridization probes showed that the 3' ends of the N and NSS mRNAs overlap each other by about 100 nucleotides. The 3' end of the NSS mRNA extends into the coding sequence of the N mRNA, whereas the N mRNA is terminated just prior to the stop codon of NSS. To our knowledge, this is the first example of overlapping complementary mRNAs in viruses with an ambisense coding strategy. No obvious transcription termination sequence was identified. However, because of a short palindromic sequence in the intergenic region, the 3' ends of both mRNAs (and consequently also the template RNAs) can be folded into an A/U-rich hairpin structure. It remains to be determined whether this structure plays any role in transcription termination.

Amino Acid Sequence↗

PubMed1998

Alterations of per RNA in noncoding regions affect periodicity of circadian behavioral rhythms.

Circadian rhythms in Drosophila depend on a molecular feedback loop that includes products of the period (per) and timeless (tim) genes. RNA and protein products of both genes cycle with a circadian period and the proteins feedback to inhibit expression of their own mRNAs. While cyclic expression of PER protein appears to be necessary for rhythmic behavior, the function of per RNA cycling is somewhat controversial. Rhythmic transcription accounts, in part, for cycling of per RNA, but it is clear now that posttranscriptional mechanisms also contribute to the cyclic expression of both per RNA and protein. As posttranscriptional mechanisms, such as mRNA stability and translation, are frequently mediated by 3' untranslated regions (UTR) of genes, the authors examined the role of this region of per in the regulation of circadian rhythms. Removal of most of per's 3' UTR had a small effect on the function of a per transgene. However, replacement of per's 3'UTR with corresponding sequences of the tubulin gene led to the rescue of behavioral rhythms in per01 flies with periods that were 3 h shorter than those generated by a wild-type per transgene. The hybrid RNA cycles, but the protein produced by it accumulates earlier in a day-night cycle than the PER protein produced by a control per transgene carrying its own 3'UTR, perhaps because the tubulin sequences counteract the effect of destabilizing elements in the per RNA at earlier points in the circadian cycle. These data indicate that the appropriate regulation of per RNA expression, effected by transcriptional as well as posttranscriptional mechanisms, is critical for the determination of circadian period.

Animals↗

PubMedDate not supplied

Renal pathology in WAGR syndrome.

The Wilms' tumor-aniridia-genital anomalies-mental retardation (WAGR) syndrome is associated with an increased risk for developing Wilms' tumor. A right nephrectomy was performed following the diagnosis of Wilms' tumor in a 2-year-old girl with WAGR syndrome and chromosome 11, del 11p13. Pathologic examination revealed intralobar nephrogenic rests and a peripelvic multicystic mass, sharply delineated from the adjacent typical intralobar nephrogenic rests and renal parenchyma, which may represent a cystic Wilms' tumor (cystic partially differentiated nephroblastoma). We studied the expression of the H19 gene by in-situ hybridization performed on paraffin sections of the kidney. H19 is an imprinted maternally-expressed gene that is not translated to protein and functions as a regulatory RNA molecule. It is tightly linked with the paternally-imprinted gene of insulin-like growth factor 2. While IGF2 presumably plays a role in tumorigenesis of Wilms' tumor, H19 is not expressed in the majority of Wilms' tumors. The expression of H19 in the intralobar nephrogenic rests was found to be prominent in the component of the blastema and markedly reduced with differentiation to tubular structures similar to the fetal kidney. The differential diagnosis of hyperplastic intralobar nephrogenic rests from a small Wilms' tumor arising in intralobar nephrogenic rests is difficult. Complete understanding of the chain of molecular events occurring in the evolution of Wilms' tumors may lead to the development of tumor markers to be used on paraffin sections and so help in the differential diagnosis of hyperplasia versus malignant transformation.

Child, Preschool↗

PubMed1991

Pretranslational mechanisms determine the type of potassium channels expressed in the rat skeletal and cardiac muscles.

We have cloned a cDNA (RMK2) coding for a Shaker type delayed rectifier K+ channel from a rat skeletal muscle cDNA library. The clone encodes a putative protein of 602 amino acids, identical with a rat brain K+ channel Kv1 (Swanson, R., Marshall, R., Smith, J. S., Williams, J. B., Boyle, M. B., Folander, K., Luneau, C. J., Antanavage, J., Oliva, C., Burhow, S. A., Bennet, C., Stein, R. B., and Kaczmarek, L. K. (1990) Neuron 4, 929-939). Northern blot analysis showed that RMK2 is expressed in skeletal and cardiac muscle. RNase protection analysis showed that the 3'-noncoding regions of the brain, cardiac, and skeletal muscle RMK2 transcripts are identical. Cloning of the gene confirmed that the protein is encoded by a single exon (Swanson et al. (1990) Neuron 4, 929-939). We expressed RMK2 in Xenopus oocytes and showed that it encodes noninactivating delayed rectifier K+ channels, resistant to block by external tetraethylammonium, with a small unitary conductance of 8.0 picosiemens. Coinjection of RMK2 and RCK1 (RMK1) (Baumann, A., Grupe, A., Ackermann, A., and Pongs, O. (1988) EMBO J. 7, 2457-2463; Koren, G., Liman, E. R., Logothetis, D. E., Nadal-Ginard, B., and Hess, P. (1990) Neuron 4, 39-51) into Xenopus oocytes resulted in the expression of currents that have tetraethylammonium inhibition curves that differ from the linear combination of inhibition curves of the two types expressed individually. Thus, RMK2 and RCK1 (RMK1) can form heteromultimers. RNA blot hybridization analysis revealed that the RMK2 transcript is developmentally regulated in a different manner in the rat skeletal muscle, ventricle, and atrium.

Aging↗

PubMed1990

Molecular biology of macrophage colony-stimulating factor.

In this chapter we have described one of the more complex hemopoietic factors, M-CSF. The single-copy M-CSF gene is almost 21 kb in length and is arranged into 10 exons and 9 introns. Expression of the gene at the RNA level is heterogeneous, and several species of M-CSF mRNA have been found in human and murine cells and tissues. In human cells the different mRNAs arise from alternative splicing of the nuclear RNA precursor in both coding and noncoding regions. This results in mRNAs encoding two distinct M-CSF proteins, 256 and 554 amino acids in length. In murine cells only a 552-amino-acid form has been found thus far. All forms of M-CSF have a 32-amino-acid signal peptide and a 23-amino-acid hydrophobic region near the carboxy-terminus, which resembles a transmembrane domain. A large portion of the carboxy-terminal end, including the hydrophobic region, is not found in the mature protein. Thus, the primary translation product of M-CSF is a prepropolypeptide, with processing occurring at both amino- and carboxy-terminal ends. The exact size of the mature protein is still somewhat in doubt, but deletion mutagenesis from the carboxy-terminal end indicates that the protein may be as small as 150 amino acids and still be functional. Site-directed mutagenesis has also shown that the first seven cysteines in the mature molecule are probably necessary for biological activity, whereas the next two cysteine residues are not. In spite of the heavy glycosylation found in the native protein, removal of the N-linked glycosylation signals does not seem to affect activity to any great degree. The M-CSF gene and its receptor, C-FMS, are tightly linked on the long arm of chromosome 5, a unique finding in the ligand/receptor field. This region also contains the genes for GM-CSF, IL-3, ECGF, and the receptor for PDGF. A similar situation may exist on chromosome 11 of the mouse. The close linkage of these factors and receptors is the probable cause for the disorders of hemopoiesis that arise when deletions occur in this area. The preceding discussion has shown how quickly the area of M-CSF molecular biology has advanced in the past 2-3 years. A great deal of effort is now being directed toward expressing M-CSF at high levels in a variety of prokaryotic and eukaryotic systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1998

Spontaneous and engineered deletions in the 3' noncoding region of tick-borne encephalitis virus: construction of highly attenuated mutants of a flavivirus.

The flavivirus genome is a positive-strand RNA molecule containing a single long open reading frame flanked by noncoding regions (NCR) that mediate crucial processes of the viral life cycle. The 3' NCR of tick-borne encephalitis (TBE) virus can be divided into a variable region that is highly heterogeneous in length among strains of TBE virus and in certain cases includes an internal poly(A) tract and a 3'-terminal conserved core element that is believed to fold as a whole into a well-defined secondary structure. We have now investigated the genetic stability of the TBE virus 3' NCR and its influence on viral growth properties and virulence. We observed spontaneous deletions in the variable region during growth of TBE virus in cell culture and in mice. These deletions varied in size and location but always included the internal poly(A) element of the TBE virus 3' NCR and never extended into the conserved 3'-terminal core element. Subsequently, we constructed specific deletion mutants by using infectious cDNA clones with the entire variable region and increasing segments of the core element removed. A virus mutant lacking the entire variable region was indistinguishable from wild-type virus with respect to cell culture growth properties and virulence in the mouse model. In contrast, even small extensions of the deletion into the core element led to significant biological effects. Deletions extending to nucleotides 10826, 10847, and 10870 caused distinct attenuation in mice without measurable reduction of cell culture growth properties, which, however, were significantly restricted when the deletion was extended to nucleotide 10919. An even larger deletion (to nucleotide 10994) abolished viral viability. In spite of their high degree of attenuation, these mutants efficiently induced protective immune responses even at low inoculation doses. Thus, 3'-NCR deletions represent a useful technique for achieving stable attenuation of flaviviruses that can be included in the rational design of novel flavivirus live vaccines.

Animals↗

PubMed1986

Poliovirus temperature-sensitive mutant containing a single nucleotide deletion in the 5'-noncoding region of the viral RNA.

The effect on viral replication of deleting nucleotide 10 of the poliovirus RNA genome was determined. This deletion, which removes a base pair from a predicted hairpin structure in the viral RNA, was introduced into full-length cDNA. Virus recovered after transfection of HeLa cells with the mutated cDNA contained the expected deletion and was temperature sensitive for plaque formation. Analysis of viral replication by one-step growth experiments indicated that mutant virus production at the nonpermissive temperature was at least 100 times less than that of wild type virus, and release of virus from mutant-infected cells was delayed. The synthesis of positive- and negative-strand viral RNA in mutant virus-infected cells was temperature sensitive. Virus-specific protein synthesis in mutant virus-infected cells was not temperature sensitive but occurred at a slower rate than that of wild type virus at permissive and nonpermissive temperatures. Replication of the mutant virus was sensitive to actinomycin D, in contrast to the wild type parent virus, which was resistant to the drug. Mutant virus stocks contained a small percentage of ts+ viruses that were able to form plaques at the nonpermissive temperature. Nucleotide sequence analysis of genomic RNA from these ts+ viruses revealed a single base change at position 34 from a G to U. In the positive RNA strand, the effect of this mutation is to restore to the hairpin structure the single base pair whose formation was prevented by the original deletion. The ts+ pseudorevertants replicated to similar titers as wild type virus at 33 and 38.5 degrees and were partially sensitive to actinomycin D.

Base Sequence↗

PubMed1978

Conservation of the primary structure at the 3' end of 18S rRNA from eucaryotic cells.

DNA sequencing methods have been used to determine a sequence of about 20 nucleotides at the 3' termini of various 18S (small ribosomal subunit) RNA molecules. Polyadenylated rRNA was first synthesized using the enzyme ATP:polynucleotidyl transferase from mainze. Then in the presence of an oligonucleotide primer uniquely complementary to the end of each adenylated rRNA, a cDNA copy was produced using AMV reverse transcriptase. In every case, the cDNA transcript was of finite size, which we ascribe to the appearance of an oligonucleotide containing m62A near the 3' end of the 18S rRNAs. Sequences at the 3' termini of 18S rRNA molecules from the four eucaryotic species examined here (mouse, silk worm, wheat embryo and slime mold) are highly conserved. They also exhibit strong homology to the 3' end of E. coli 16S rRNA. Two important differences, however, are apparent. First, the 16S sequence CCUCC, implicated in mRNA binding by E. coli ribosomes, is absent from each eucaryotic rRNA sequence. Second, a purine-rich region which exhibits extensive complementarity to the 5' noncoding regions of many eucaryotic mRNAs appears consistently.

Animals↗

PubMed1998

Comparative study of different standardization concepts in quantitative competitive reverse transcription-PCR assays.

Four different standardization approaches based on a competitive reverse transcription (RT)-PCR assay were compared with a noncompetitive assay based on an external standard curve. Criteria for assessment were accuracy in quantitation, correctness of recovery, sensitivity, dynamic range, reproducibility, throughput, and convenience of sample handling. As a model system, we used the 5'-noncoding region of hepatitis C virus (HCV) for amplification in all quantitative RT-PCRs. A computer program that allowed parallel data processing was developed. Surprisingly, all methods were found suitable for accurate quantitation and comparable with respect to the criterion correctness of recovery. All results differed only by a factor of about 2. The reason for this finding might be that all of our mimics, as well as the wild-type genome of HCV, exhibited exactly the same amplification and hybridization efficacy. Moreover, minimal competition occurred in our experiments over a 5-log dynamic range. A further topic of our investigation was the comparison of two different competitive RNA fragments, mimics, with regard to their suitability as internal standards. One was a heterologous mimic, in which only the primer binding sites were identical to the wild type. The second one was a homologous mimic identical to the wild type except for a small region used for differential hybridization, which was replaced by a permutated sequence of the same length. Both the homologous and heterologous internal mimics were found appropriate for an accurate competitive RT-PCR assay, provided that amplification efficacy, as well as capture efficacy, is proven identical for both analyte and mimic.

Gene Amplification↗

PubMed1983

Cloning and analysis of cDNA sequences coding for two 16 kilodalton heat shock proteins (hsps) in Caenorhabditis elegans: homology with the small hsps of Drosophila.

The nucleotide sequences of two different cDNAs, CEHS48 and CEHS41, coding for the 16,000 dalton heat shock proteins (hsps) of Caenorhabditis elegans have been determined. CEHS48 codes for a polypeptide of 135 amino acids, approximately 15 fewer than the complete protein while CEHS41 is missing approximately 46 amino acids. From nucleotide 113 to the TAA termination signal the extent of homology between the sequences is 91%. Toward the 5' ends, the homology drops to 20% and results in completely divergent amino acid sequences. The 3' noncoding regions are only 30% homologous. Only CEHS48 contains a poly(A) signal and a poly(A) tail, suggesting that CEHS41 has an incomplete 3' end. The region from amino acid 43 to amino acid 115 shows extensive homology with corresponding regions in the four small hsps of Drosophila melanogaster and in mammalian alpha-crystallin. Two-dimensional gel analysis of in vitro synthesized hsp16 reveals the existence of five distinct components of identical molecular weights, but with different isoelectric points.

Amino Acid Sequence↗

PubMed1997

Internal entry of ribosomes is directed by the 5' noncoding region of classical swine fever virus and is dependent on the presence of an RNA pseudoknot upstream of the initiation codon.

Bicistronic RNAs containing the 373-nucleotide-long 5' nontranslated region (NTR) of the classical swine fever virus (CSFV) genome as intercistronic spacer were used to show the presence of an internal ribosome entry site (IRES) in the 5' end of the CSFV genome. By coexpression of the poliovirus 2A protease it was demonstrated that the CSFV 5' NTR-driven translation is independent of the presence of functional eukaryotic initiation factor eIF-4F. Deletion analysis indicated that the 5' border of the IRES is located between nucleotides 28 and 66. The role of a proposed pseudoknot structure at the 3' end of the CSFV 5' NTR in IRES-mediated translation was investigated by site-directed mutagenesis. Mutant RNAs that had lost the ability to base pair in stem II of the pseudoknot were translationally inactive. Translation to wild-type levels could be restored through the introduction of compensatory complementary base changes that repaired base pairing in stem II. In addition, we showed that the AUG codon, which is located 7 nucleotides upstream of the polyprotein initiation site and is conserved in pestiviruses, could not be used to initiate translation. Also, an AUG codon introduced downstream of the polyprotein initiation site was not recognized as an initiation site by ribosomes. These data suggest that after internal entry on the CSFV 5' NTR, ribosomal scanning for the initiation codon is limited to a small region.

Animals↗

PubMed1982

Use of recombinant DNA technology to program eukaryotic cells to synthesize rat proinsulin: a rapid expression assay for cloned genes.

To use recombinant DNA technology to functionally analyze mutations introduced into cloned eukaryotic genes, a rapid procedure is necessary to assay the steps along the gene expression pathway. Since cloned rat insulin genes are not transcribed efficiently after transfection into various cell lines, I have asked whether one could drive expression by placing the insulin gene inside a transcriptional unit that functions in all mammalian cells. By using a small simian virus 40 (SV40) fragment that contains initiation signals for replication and transcription, I connected the 5'-noncoding region of the SV40 tumor antigen gene to the 5'-noncoding region of the rat insulin II gene to create a pBR322-based recombinant. If one assays shortly after its introduction into mammalian cells, it can be shown that this recombinant plasmid programs the synthesis of correctly spliced and polyadenylylated insulin mRNA that functions in the synthesis and secretion of rat proinsulin. This system permits rapid analysis of cloned in vitro-engineered mutations and the programming of eukaryotic cells to manufacture proteins that they normally do not synthesize.

Animals↗

PubMed1993

Evolution of hominoid mitochondrial DNA with special reference to the silent substitution rate over the genome.

Focusing on the synonymous substitution rate, we carried out detailed sequence analyses of hominoid mitochondrial (mt) DNAs of ca. 5-kb length. Owing to the outnumbered transitions and strong biases in the base compositions, synonymous substitutions in mtDNA reach rapidly a rather low saturation level. The extent of the compositional biases differs from gene to gene. Such changes in base compositions, even if small, can bring about considerable variation in observed synonymous differences and may result in the region-dependent estimate of the synonymous substitution rate. We demonstrate that such a region dependency is due to a failure to take proper account of heterogeneous compositional biases from gene to gene but that the actual synonymous substitution rate is rather uniform. The synonymous substitution rate thus estimated is 2.37 +/- 0.11 x 10(-8) per site per year and comparable to the overall rate for the noncoding region. On the other hand, the rate of nonsynonymous substitutions differs considerably from gene to gene, as expected under the neutral theory of molecular evolution. The lowest rate is 0.8 x 10(-9) per site per year for COI and the highest rate is 4.5 x 10(-9) for ATPase 8, the degree of functional constraints (measured by the ratio of the nonsynonymous to the synonymous substitution rate) being 0.03 and 0.19, respectively. Transfer RNA (tRNA) genes also show variability in the base contents and thus in the nucleotide differences. The average rate for 11 tRNAs contained in the 5-kb region is 3.9 x 10(-9) per site per year. The nucleotide substitutions in the genome suggest that the transition rate is about 17 times faster than the transversion rate.

Adenosine Triphosphatases↗

PubMed1984

Nucleotide sequence of avian carcinoma virus MH2: two potential onc genes, one related to avian virus MC29 and the other related to murine sarcoma virus 3611.

The 5.2-kilobase (kb) RNA genome of avian carcinoma virus MH2 has the genetic structure 5'-delta gag (0.2 kb)- mht (1.2 kb)-myc (1.4 kb)-c (0.4 kb)-poly(A) (0.2 kb)-3'. delta gag is a partial retroviral core protein gene, mht and myc are cell-derived MH2-specific sequences, and c is the 3'-terminal retroviral vector sequence. Here we have determined the nucleotide sequence of 3.5 kb from the 3' end of delta gag to the 3' end of molecularly cloned proviral MH2 DNA, in order to elucidate the genetic structure of the virus and to compare it with other mht - and myc-containing oncogenic viruses as well as with the chicken proto-myc gene. The following results were obtained: (i) delta gag- mht forms a hybrid gene with a contiguous reading frame of 2682 nucleotides that terminates with a stop codon near the 3' end of mht . The 3' 969 nucleotides of mht up to the stop codon are 80% sequence related to the onc-specific raf sequence of murine sarcoma virus 3611 (94% homologous at the deduced amino acid level). (ii) The myc sequence is preceded by an RNA splice acceptor site shared with the cellular proto-myc gene, beyond which it is colinear up to a 3'-termination codon and 40 noncoding nucleotides with the myc sequences of avian retrovirus MC29 and chicken proto-myc. Thus, myc forms, together with a 5' retroviral exon, a second MH2-specific gene. (iii) myc is followed by the 3'-terminal c region of about 400 nucleotides, which is colinear with that of Rous sarcoma virus except for a substitution near the 5' end of the long terminal repeat. It is concluded that MH2 contains two genes with oncogenic potential, the delta gag- mht gene, which is closely related to the delta gag-raf transforming gene of MSV 3611, and the myc gene, which is related to the transforming gene of MC29. Furthermore, it may be concluded that the cellular proto-onc genes, which on sequence transduction become viral onc genes, are a small group because among the 19 known onc sequences, 5 are shared by different taxonomic groups of viruses of which the mht /raf homology is the closest determined so far.

Amino Acid Sequence↗

PubMed1992

The small genome of Arabidopsis contains at least nine expressed beta-tubulin genes.

The small genome of Arabidopsis contains at least nine expressed beta-tubulin (TUB) genes, in contrast to the large genomes of vertebrate animals, which contain a maximum of seven expressed beta-tubulin genes. In this study, we report the structures of seven new TUB genes (TUB2, TUB3, TUB5, TUB6, TUB7, TUB8, and TUB9) of Arabidopsis. The sequences of TUB1 and TUB4 had been reported previously. Sequence similarities and unique structural features suggest that the nine TUB genes evolved by way of three branches in the plant beta-tubulin gene evolutionary tree. Two genes (TUB2 and TUB3) encode the same beta-tubulin isoform; thus, the nine genes predict eight different beta-tubulins. In contrast to the alpha-tubulin (TUA) genes with their divergent intron patterns, all nine TUB genes contain 2 introns at conserved positions. Noncoding 3' gene-specific hybridization probes have been constructed for all nine TUB genes and used in RNA gel blot analyses to demonstrate that all nine genes are transcribed. Two-dimensional protein immunoblot analyses have resolved at least seven different beta-tubulin isoforms in Arabidopsis, indicating that most, if not all, of the TUB transcripts are translated.

Amino Acid Sequence↗

PubMed1989

Genetic variation occurring on the genome of an in vitro insertion mutant of poliovirus type 1.

An insertion sequence of 72 nucleotides prepared from a polylinker sequence of plasmid pUC18 was introduced at nucleotide position 702 of the 5' noncoding sequence (742 nucleotides long) of the genome of the Sabin strain of poliovirus type 1 by using an infectious cDNA clone of the virus strain. The insertion mutant thus obtained showed a small-plaque phenotype compared with that of the parent virus. Apparent revertants (large-plaque variants) were easily generated from the insertion mutant. Nucleotide sequence analysis was performed on the revertant genomes to determine the mutation(s) by which the plaque size of the parent virus was regained. Some large-plaque variants lacked genomic sequences including all or a part of the insertion sequence. A computer-aided search for secondary structures with respect to the deletion sites detected possible supporting sequences which provided fairly stable secondary structures at the deletion sites. This result was consistent with our supporting sequence-loop model which had been proposed as a new copy-choice model for the generation of genetic rearrangements occurring on single-stranded RNA genomes (S. Kuge, I. Saito, and A. Nomoto, J. Mol. Biol. 192:473-487, 1986). The other large-plaque variants had point mutations at any one of three positions of an AUG existing in the insertion sequence. A small-plaque phenotype was observed when an AUG codon was inserted in frame or out of frame with regard to the initiation site of viral polyprotein synthesis. Our data strongly suggest that an AUG sequence in this genome region is deleterious for efficient poliovirus replication.

Chromosome Deletion↗

PubMed1987

Origin of the human L1 elements: proposed progenitor genes deduced from a consensus DNA sequence.

A consensus sequence for the human long interspersed repeated DNA element, L1Hs (LINE or KpnI sequence), is presented. The sequence contains two open reading frames (ORFs) which are homologous to ORFs in corresponding regions of L1 elements in other species. The L1Hs ORFs are separated by a small evolutionarily nonconserved region. The 5' end of the consensus contains frequent terminators in all three reading frames and has a relatively high GC content with numerous stretches of weak homology with AluI repeats. The 5' ORF extends for a minimum of 723 bp (241 codons). The 3' ORF is 3843 bp (1281 codons) and predicts a protein of 149 kD which has regions of weak homology to the polymerase domain of various reverse transcriptases. The 3' end of the consensus has a 208-bp nonconserved region followed by an adenine-rich end. The organization of the L1Hs consensus sequence resembles the structure of eukaryotic mRNAs except for the noncoding region between ORFs. However, due to base substitutions or truncation most elements appear incapable of producing mRNA that can be translated. Our observation that individual elements cluster into subfamilies on the basis of the presence or absence of blocks of sequence, or by the linkage of alternative bases at multiple positions, suggests that most L1 sequences were derived from a small number of structural genes. An estimate of the mammalian L1 substitution rate was derived and used to predict the age of individual human elements. From this it follows that the majority of human L1 sequences have been generated within the last 30 million years. The human elements studied here differ from each other, yet overall the L1Hs sequences demonstrate a pattern of species-specificity when compared to the L1 families of other mammals. Possible mechanisms that may account for the origin and evolution of the L1 family are discussed. These include pseudogene formation (retroposition), transposition, gene conversion, and RNA recombination.

Biological Evolution↗