Mapping unintegrated avian sarcoma virus DNA: termini of linear DNA bear 300 nucleotides present once or twice in two species of circular DNA.

Three major species of viral DNA have been observed in cells infected by retroviruses: a linear, double-stranded copy of a subunit of viral RNA; closed circular DNA; and proviral DNA inserted covalently into the genome of the host cell. We have studied the structures of the unintegrated forms of avian sarcoma virus (ASA) DNA using agarose gel electrophoresis in conjunction with restriction endonucleases and molecular hybridization techniques. The linear duplex DNA is approximately the same length as a subunit of viral RNA (approximately 10 kb) and it bears natural repeats of approximately 300 nucleotides at its termini. The repeats are composed of sequences derived from both the 3' and 5' termini of viral RNA in a manner suggesting that the viral DNA polymerase is transferred twice between templates. Thus the first end begins with a sequence from the 5' terminus of viral RNA and is permuted by about 100 nucleotides with respect to the 3' terminus of viral RNA; the linear DNA terminates with a sequence of about 200 nucleotides derived from the 3' end of viral RNA. We represent this structure, synthesized from right to left, as 3'5'-----3'5'. Two closed circular species of approximately monomeric size have been identified. The less abundant species contain all the sequences identified in linear DNA, including two copies in tandem of the 300 nucleotide 3'5' repeat. The major species lacks about 300 base pairs (bp) mapped to the region of the repeated sequence; thus it presumably contains only a single copy of that sequence. The strategies used to determine these structures involved the assignment of over 20 cleavage sites for restriction endonucleases on the physical maps of ASV DNA. Several strains of ASV were compared with respect to these sites, and the sites have been located in relation to deletions frequently observed in the env and src genes of ASV.     

Extreme ends of the genome are conserved and rearranged in the defective interfering RNAs of semliki forest virus

We have analyzed Semliki Forest virus defective interfering RNA molecules, generated by serial undiluted passaging of the virus in baby hamster kidney cells. The 42 S RNA genome (about 13 kb ²) has been greatly deleted to generate the DI RNAs, which are heterogeneous both in size (about 2 kb) and sequence content. The DI RNAs offer a system for exploring binding sites for RNA polymerase and encapsidation signals, which must have been conserved in them since they are replicated and packaged. In order to study the structural organization of DI RNAs, and to analyze which regions from the genome have been conserved, we have determined the nucleotide sequences of (1) a 2.3 kb long DI RNA molecule, DI309, (2) 3′-terminal sequences (each about 0.3 kb) of two other DI RNAs, and (3) the nucleotide sequence of 0.4 kb at the extreme 5′ end of the 42 S RNA genome.The DI309 molecule consists of a duplicated region with flanking unique terminal sequences. A 273-nucleotide sequence is present in four copies per molecule. The extreme 5′-terminal nucleotide sequence of the 42 S RNA genome is shown to contain domains that are conserved in the two DI RNAs of known structure: DI309, and the previously sequenced DI301 (Lehtovaara et al., 1981). Here we report which terminal genome sequences are conserved in the DI RNAs, and how they have been modified, rearranged or amplified.     

Characterization of two mRNA species encoding human estradiol 17 beta-dehydrogenase and assignment of the gene to chromosome 17

Using two 33-mer synthetic oligonucleotides derived from the amino acid sequence of the catalytic site of estradiol 17 beta-dehydrogenase (E2DH) and polyclonal antibodies raised against the enzyme purified from human placenta, clones were isolated from a lambda gt11 human placental cDNA library. A 327-amino acid sequence was deduced from cDNA sequencing. Two mRNA species have been identified in poly(A)+ RNA from human placenta, a major species migrating at 1.3 kb while a minor one is found at approx. 2.2 kb. Primer extension and S1 nuclease analysis indicate that the major mRNA species starts 9-10 nucleotides while the minor mRNA starts 971 nucleotides upstream from the ATG initiating codon, respectively. Sequence analysis of the longest cDNA clone (2092 bp) shows that it possesses identical coding and non-coding sequences in the regions of overlap with the shorter cDNA clones. The 32P-labeled 5' non-coding fragment hybridizes only to the 2.2 kb band, thus providing evidence for the existence of two distinct mRNA species which differ in their 5' noncoding regions. Using hp E2DH-36 cDNA as a probe for in situ hybridization of translocated chromosomes, the human E2DH gene was localized to the q11-q12 region of chromosome 17.     

The Bos taurus casein genes. Isolation and characterization of the kappa-casein gene

A region spanning 25 kb of genomic DNA containing the kappa-casein gene, has been isolated from two genomic libraries in EMBL3 and EMBL4 phage vectors. Five phage clones containing kappa-casein gene have been found. Gene organisation has been determined using restriction mapping and a partial sequencing the 5' and 3' flanking regions. The kappa-casein gene includes 5 exons, the first of them coding for 64 nucleotides from the 5' untranslated mRNA zone. The gene is 12.5 kb long, which is almost 16 times longer than the corresponding mRNA. The first intron spans 2.5 kb, the second is the largest one and spans 5.5 kb. The 5' flanking region sequence has been analysed; it contains a TATA box from -30 to -25 bp, somewhat different from the canonic sequence, and a CAAT box at -80 bp.     

Binding of ribosomes to the 5' leader sequence (N = 258) of RNA 3 from alfalfa mosaic virus.

RNA 3 of alfalfa mosaic virus (AlMV) contains information for two genes: near the 5' end an active gene coding for a 35 Kd protein and, near the 3' end, a silent gene coding for viral coat protein. We have determined a sequence of 318 nucleotides which contains the potential initiation codon for the 35 Kd protein at 258 nucleotides from the 5' end. This long leader sequence can form initiation complexes containing three 80 S ribosomes. A shorter species of RNA, corresponding to a molecule of RNA 3 lacking the cap and the first 154 nucleotides (RNA 3') has been isolated. The remaining leader sequence of 104 nucleotides in RNA 3' forms a single 80 S initiation complex with wheat germ ribosomes. The location of the regions of the leader sequence of RNA 3 involved in initiation complex formation with 80 S ribosomes is reported.     

Analysis of DNA encoding 23S rRNA and 16S–23S rRNA intergenic spacer regions from Plesiomonas shigelloides

Amplification of the gene encoding 23S rRNA of Plesiomonas shigelloides by polymerase chain reaction (PCR), with primers complementary to conserved regions of 16S and the 3' end of 23S rRNA genes, resulted in a DNA fragment of approximately 3 kb. This fragment was cloned in Escherichia coli and its nucleotide sequence determined. The region encoding 23S rRNA shows high homology with the published sequences of 23S rRNA from other members of the gamma division of Proteobacteria. The sequence of the intergenic spacer region, between the 16S and 23S rRNA genes, was determined in a further two clones. In one the sequence of a single tRNA(Glu) was found which was absent from the other two. This variation in sequence suggests that the different clones may be derived from different ribosomal RNA operons.     

Heteroduplex study of the sequence relations between RD-114 and baboon viral RNAs.

The regions of sequence homology and nonhomology between the RNA genomes of RD-114 and baboon endogenous type C viruses have been mapped by an electron microscope heteroduplex study. Short complementary DNA (cDNA) copies (approximately 150 to 200 nucleotides in length) of RD-114 RNA were prepared by an endogenous synthesis; labels of polydeoxythymidylic acid [poly(dT)] were attached to the 3' ends of the cDNA molecules by a reaction catalyzed by deoxynucleotidyl terminal transferase. The cDNA-poly(dT) was hybridized to RD-114 RNA and to baboon viral RNA dimer (50 to 70S) units, and the position- of the poly(dT) labels were observed by electron microscopy. With RD-114, labels were distributed uniformly along the genome. With baboon virus RNA (monomer length, 9.5 kilobases [kb]), the regions of high homology with RD-114 cDNA were observed to lie in the intervals from 1.5 to 2.5 kb and from 3.7 to 5.5 kb from the 5' end. The relations of these heteroduplex maps to the known antigenic similarities and differences among the several viral proteins and to the genetic maps of the viruses are discussed.     

Molecular structure and flanking nucleotide sequences of the natural chicken ovomucoid gene

Five independent clones containing the natural chicken ovomucoid gene have been isolated from a chicken gene library. One of these clones, CL21, contains the complete ovomucoid gene and includes more than 3 kb of DNA sequences flanking both termini of the gene. Restriction endonuclease mapping, electron microscopy and direct DNA sequencing analyses of this clone have revealed that the ovomucoid gene is 5.6 kb long and codes for a messenger RNA of 821 nucleotides. The structural gene sequence coding Ifor the mature messenger RNA is split into at least eight segments by a minimum of seven intervening sequences of various sizes. The shortest structural gene segment is only 20 nucleotides long. All seven intervening sequences are located within the peptide coding region of the gene, and the sequences at the 5' and 3' untranslated regions of the mRNA are not interrupted by intervening sequences. The DNA sequences of the regions flanking the 5' and 3' termini of the gene have been determined. Thirty nucleotides before the start of the messenger RNA coding sequence is the heptanucleotide TATATAT, which is also present in a similar location relative to the chicken ovalbumin gene and other unique sequence eucaryotic genes. This sequence resembles that of the Pribnow box in procaryotic genes where a promoter function has been implicated. Seven nucleotides past the 3' end of the gene is the tetranucleotide TTGT, a sequence found to be present at identical locations as either TTTT or TTGT in other eucaryotic genes that have been sequenced. These conserved DNA sequences flanking eucaryotic genes may serve some regulator function in the expression of these genes.     

Deletion mapping of a mouse hepatitis virus defective interfering RNA reveals the requirement of an internal and discontiguous sequence for replication.

All of the defective interfering (DI) RNAs of mouse hepatitis virus (MHV) contain both the 5' and 3' ends of the viral genomic RNA, which presumably include the cis sequences required for RNA replication. To define the replication signal of MHV RNA, we have used a vaccinia virus-T7 polymerase-transcribed MHV DI RNA to study the effects of sequence deletion on DI RNA replication. Following infection of susceptible cells with a recombinant vaccinia virus expressing T7 RNA polymerase, various cDNA clones derived from a DI RNA (DIssF) of the JHM strain of MHV, which is a 3.5-kb naturally occurring DI RNA, behind a T7 promoter were transfected. On superinfection with a helper MHV, the ability of various DI RNAs to replicate was determined. Serial deletions from the middle of the RNA toward both the 5' and 3' ends demonstrated that 859 nucleotides from the 5' end and 436 nucleotides from the 3' end of the MHV RNA genome were necessary for RNA replication. Surprisingly, an additional stretch of 135 nucleotides located at 3.1 to 3.3 kb from the 5' end of the genome was also required. This stretch is discontiguous from the 5'-end cis replication signal and is present in all of the naturally occurring DI RNAs studied so far. The requirement for a long stretch of 5'- and 3'-end sequences predicts that the subgenomic MHV mRNAs cannot replicate. The efficiency of RNA replication varied with different cDNA constructs, suggesting possible interaction between different regions of DI RNA. The identification of MHV RNA replication signals allowed the construction of an MHV DI-based expression vector, which can express foreign genes, such as the chloramphenicol acetyltransferase gene.     

A mutation which alters initiation of transcription by RNA polymerase III on the Ad5 chromosome

Mutant dl 309 is a viable Ad5 deletion mutant. Whereas wild-type Ad5-infected HeLa cells contain two VAI RNA species [VAI(A) and VAI(G)] which differ by three nucleotides at their 5' ends, dl 309-infected HeLa cells contain VAI(G) but no VAI(A) RNA. Nucleotide sequence analysis indicates that dl 309 lacks two base pairs which precede the 5' end of VAI(A) by 22 nucleotides. Since the 5' ends of VAI RNAs are not processed, the 309 deletion serves to identify a portion of the sequence required for RNA polymerase III initiation. Since dl 309 grows as well as wild-type Ad5 in HeLa cells, the VAI(A) species is not essential for viral growth in these cells.     

Sequence organization of feline leukemia virus DNA in infected cells

A restriction site map has been deduced of unintegrated and integrated FeLV viral DNA found in human RD cells after experimental infection with the Gardner-Arnstein strain of FeLV. Restriction fragments were ordered by single and double enzyme digests followed by Southern transfer (1) and hybridization with 32P-labeled viral cDNA probes. The restriction map was oriented with respect to the 5' and 3' ends of viral RNA by using a 3' specific hybridization probe. The major form of unintegrated viral DNA found was a 8.7 kb linear DNA molecule bearing a 450 bp direct long terminal redundancy (LTR) derived from both 5' and 3' viral RNA sequences. Minor, circular forms, 8.7 kb and 8.2 kb in length were also detected, the larger one probably containing two adjacent copies of the LTR and the smaller one containing one comtaining one copy of the LTR. Integrated copies of FeLV are colinear with the unintegrated linear form and contain the KpnI and SmaI sites found in each LTR.     

大麦条纹花叶病毒新疆株3''''端基因组的克隆及分析

我们应用多次加尾的方法,对大麦条纹花叶病毒新疆株的RNAs基因组进行克隆。在所述条件下可以合成2000～3000核苷酸长度的cDNA。通过原位杂交、转印杂交(Northern blot)证明所得克隆属于RNA_1和RNA_3组分,并包含有3′端基因组结构。对部分克隆进行了酶谱分析。     

Effects of cDNA hybridization on translation of encephalomyocarditis virus RNA.

Cell-free translation of the RNA of encephalomyocarditis virus was examined after hybridization of chemically synthesized cDNA fragments to different sites of the 5' noncoding region of the viral RNA. The following results were obtained. The binding of cDNA fragments to the first 41 nucleotides, to the poly(C) tract (between nucleotides 149 and 263), and to the sequence between nucleotides 309 and 338 did not affect translation of the viral RNA; the binding of cDNA fragments to the sequence between nucleotides 420 and 449 caused a slight inhibition; and the binding of fragments to eight different sites between nucleotides 450 and the initiator AUG codon (nucleotide 834) caused high degrees of inhibition. The results suggest that the first part of the 5' untranslated region, at least to nucleotide 338, may not be required for encephalomyocarditis viral RNA translation; however, the region near nucleotide 450 is important for translation of the viral RNA. The possibility that initiation occurs at an internal site is discussed.     

Isolation of cDNA clones coding for mitochondrial 16S ribosomal RNA from the crustacean Artemia

cDNA clones coding for Artemia mitochondrial 16S ribosomal RNA (rRNA) have been isolated. The clones cover from nucleotide 650 of the RNA molecule to its 3' end. The comparison of Artemia sequence with both vertebrate and invertebrate mitochondrial 16S rRNA sequences has shown the existence of regions of high similarity between them. A model for the secondary structure of the 3' half of Artemia mitochondrial 16S rRNA is proposed. The size of the rRNA molecule has been estimated at 1.35 kb. Despite the similarity of the Artemia gene to insect rRNA in size, sequence and secondary structure, the G + C content of the Artemia gene (42%) is closer to that of mammals than to the insect genes. The number of mitochondria in Artemia has been estimated at 1500 per diploid genome in the cyst and 4000 in the nauplius. In contrast, the amount of mt 16S rRNA is constant at all stages of Artemia development.     

Specific binding of host cellular proteins to multiple sites within the 3' end of mouse hepatitis virus genomic RNA.

The initial step in mouse hepatitis virus (MHV) RNA replication is the synthesis of negative-strand RNA from a positive-strand genomic RNA template. Our approach to begin studying MHV RNA replication is to identify the cis-acting signals for RNA synthesis and the proteins which recognize these signals at the 3' end of genomic RNA of MHV. To determine whether host cellular and/or viral proteins interact with the 3' end of the coronavirus genome, an RNase T1 protection/gel mobility shift electrophoresis assay was used to examine cytoplasmic extracts from mock- and MHV-JHM-infected 17Cl-1 murine cells for the ability to form complexes with defined regions of the genomic RNA. We demonstrated the specific binding of host cell proteins to multiple sites within the 3' end of MHV-JHM genomic RNA. By using a set of RNA probes with deletions at either the 5' or 3' end or both ends, two distinct binding sites were located. The first protein-binding element was mapped in the 3'-most 42 nucleotides of the genomic RNA [3' (+42) RNA], and the second element was mapped within an 86-nucleotide sequence encompassing nucleotides 171 to 85 from the 3' end of the genome (171-85 RNA). A single potential stem-loop structure is predicted for the 3' (+)42 RNA, and two stem-loop structures are predicted for the 171-85 RNA. Proteins interacting with these two elements were identified by UV-induced covalent cross-linking to labeled RNAs followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The RNA-protein complex formed with the 3'-most 42 nucleotides contains approximately five host polypeptides, a highly labeled protein of 120 kDa and four minor species with sizes of 103, 81, 70, and 55 kDa. The second protein-binding element, contained within a probe representing nucleotides 487 to 85 from the 3' end of the genome, also appears to bind five host polypeptides, 142, 120, 100, 55, and 33 kDa in size, with the 120-kDa protein being the most abundant. The RNA-protein complexes observed with MHV-infected cells in both RNase protection/gel mobility shift and UV cross-linking assays were identical to those observed with uninfected cells. The possible involvement of the interaction of host proteins with the viral genome during MHV replication is discussed.     

Sequence analysis of cDNA''s derived from the RNA of Sindbis virions and of defective interfering particles

Sindbis virus generates defective interfering (DI) particles during serial high-multiplicity passage in cultured cells. These DI particles inhibit the replication of infectious virus and can be an important factor in the establishment and maintenance of persistent infection in BHK cells. In an effort to understand how these DI particles are generated and how they interfere with the replication of standard virus, we performed a partial sequence analysis of the RNA obtained from two independently isolated populations of DI particles and from two Sindbis virus variants and compared these with the RNA of the parental wild-type virus. The 3'-terminal regions of the RNAs were sequenced by the dideoxy chain terminating method. Internal regions of the RNA were examined by restriction endonuclease digestion of cDNA's made to the various RNAs and by direct chemical sequencing of 5' end-labeled restriction fragments from cDNA made to the DI RNAs. One of the variant viruses examined was originally derived from cells persistently infected with Sindbis virus for 16 months and is resistant to interference by the DI strains used. In the 3'-terminal region of the RNA from this variant, only two base changes were found; one of these occurs in the 20-nucleotide 3'-terminal sequence which is highly conserved among alphaviruses. The DI RNA sequences were found to have been produced not by a single deletional event, but by multiple deletion steps combined with sequence rearrangements; all sequences examined are derived from the plus strand of Sindbis virion RNA. Both DI RNAs had at least 50 nucleotides of wild-type sequence conserved at the 3' terminus; in addition, they both contained conserved and perhaps amplified sequences derived from the non-26S region of the genome which may be of importance in their replication and interference ability.     

Zoogloea ramigera: A phylogenetically diverse species

Abstract Amplification of the gene encoding 23S rRNA of Aeromonas hydrophila by polymerase chain reaction, with primers complementary to conserved regions of 16S and the 3'-end of 23S rRNA genes, resulted in a DNA fragment of approximately 3 kb. This fragment was cloned in Escherichia coli , and its nucleotide sequence determined. The region encoding 23S rRNA shows high homology with the published sequences of 23S rRNA from other members of the gamma division of Proteobacteria . The sequence of the intergenic spacer region, between the 16S and 23S rRNA genes, was determined in five clones. Three types of spacer were identified: two clones were identical and encoded tRNA^Ile and tRNA^Ala while the remaining three clones contained tRNA^Glu, only two had the same spacer sequences. This variation in sequence indicates that the different clones may be derived from different ribosomal RNA operons.