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PubMed1999

Splicing-independent processing of plant box C/D and box H/ACA small nucleolar RNAs.

Small nucleolar RNAs (snoRNAs) are involved in various aspects of ribosome biogenesis and rRNA maturation. Plants have a unique organisation of snoRNA genes where multiple, different genes are tightly clustered at a number of different loci. The maize gene clusters studied here include genes from both of the two major classes of snoRNAs (box C/D and box H/ACA) and are transcribed as a polycistronic pre-snoRNA transcript from an upstream promoter. In contrast to vertebrate and yeast intron-encoded snoRNAs, which are processed from debranched introns by exonuclease activity, the particular organisation of plant snoRNA genes suggests a different mode of expression and processing. Here we show that single and multiple plant snoRNAs can be processed from both non-intronic and intronic transcripts such that processing is splicing-independent and requires endonucleolytic activity. Processing of these different snoRNAs from the same polycistronic transcript suggests that the processing machineries needed by each class are not spatially separated in the nucleolus/nucleus.

Base Sequence↗
PubMed1990

The intron of the yeast actin gene contains the promoter for an antisense RNA.

Using Northern blot analysis we have detected an approximately 840 nucleotide-long RNA which is complementary to the 5' leader sequence and the first ten nucleotides of the coding sequence of the yeast actin (ACT1) messenger RNA. We have determined two transcription start sites for this actin antisense RNA (ASR1), both within the ACT1 intron, at about 80 and 90 nucleotides downstream from the 5' splice site. Analysis of a cDNA clone showed that this RNA species overlaps the entire trailer sequence and approximately 20 nucleotides of the coding sequence of the nearby yeast YPT1 gene.

Actins↗
PubMed1984

Autoantibodies reactive with small ribonucleoprotein antigens: a convergence of molecular biology and clinical immunology.

Autoantibodies to nuclear antigens (ANA) occur in patients with systemic lupus erythematosus (SLE) and other multisystem autoimmune diseases. Although heterogeneous, there are 2 major groups, autoantibodies to DNA and autoantibodies to non-DNA antigens, the latter including ANAs to the soluble or "extractable nuclear antigens" (ENA). This review discusses those ENAs which are ribonucleoproteins (RNPs) consisting of small RNA molecules (80-400 nucleotides) attached to non-histone proteins: these are called small nuclear (sn) or small cytoplasmic (sc) ribonucleoproteins according to their location in the cell and at least some are known to play an important role in nuclear metabolism. ENAs can be immunoprecipitated from crude preparations of nuclei by sera from patients with multisystem autoimmune diseases and, after removal of the associated proteins, the RNA components can be analyzed by gel electrophoresis. This shows 3 main categories of small RNAs: the U group comprising U1-U6 snRNAs, the Ro group comprising small nucleocytoplasmic RNAs, and the La group comprising several species of cellular snRNAs as well as the Ro scRNAs. La, in addition, includes small RNAs encoded by adenovirus (VA I, VA II), Epstein-Barr virus (EBER 1, EBER 2) and vesicular stomatitis virus (leader RNA). In the case of each group, the RNAs themselves are not antigenic but become so when associated with proteins, most of which are uncharacterized. The U snRNAs, located in the nucleus, are transcribed by RNA polymerase II and appear to be involved in the splicing of introns from mRNA. Sera from patients with mixed connective tissue disease (MCTD) react with RNPs containing U1 RNA and sera from patients with SLE react with U RNPs containing U1, U2, U4, U5 and U6 RNAs, collectively known as the Sm antigen. The Ro RNAs are transcribed by RNA polymerase III and have no known function. Sera from patients with primary Sjögren's syndrome and some cases of SLE react with Ro scRNPs. The La RNAs are also transcribed by RNA polymerase III and are located mostly in the nucleus; functionally the protein associated with the La RNAs appears to be important in RNA polymerase III transcription. Sera from patients with primary Sjögren's syndrome react with the heterogeneous group of both cellular and viral RNAs which constitute the La RNP antigen. Sera of patients with SLE, scleroderma, polymyositis and dermatomyositis also react with RNPs relevant to nuclear metabolism and further definition of these RNPs is awaited. Many advances can be expected from the convergence of molecular biology and clinical immunology exemplified by the current studies on ENAs.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Antinuclear↗
PubMed1991

Simian virus 40 late mRNA leader sequences involved in augmenting mRNA accumulation via multiple mechanisms, including increased polyadenylation efficiency.

We have examined the contribution of 5' leader sequences to expression directed by the simian virus 40 (SV40) late promoter. These studies showed that addition of sequences which contain the late leader 3' splice site to the late promoter led to an increase in the accumulation of mRNA expressed by the promoter. No other sequences within the leader region, between SV40 positions 334 and 560, exhibited a substantial influence on mRNA accumulation. The increase was due, at least in part, to the creation of a spliceable mRNA transcript, since mutation of either the 5' or 3' splice site could attenuate the effect. However, sequences at or near the 3' splice site appeared to play a more important role than did the 5' splice site in bringing about this increase. In many instances, mutation of the 3' splice site also led to the accumulation of extended transcripts, whereas mutation of the 5' splice site did not produce this result in any instance. Analysis of these extended transcripts showed that they retained sequences normally lost upon cleavage and polyadenylation. This finding suggested that mutation of the 3' splice site sequence led to decreases in the efficiency of polyadenylation. We propose that the SV40 late leader sequences positively contribute to expression of the viral late genes by increasing mRNA accumulation via multiple mechanisms, including the enhancement of pre-mRNA polyadenylation efficiency.

Animals↗
PubMed1990

All subgenomic mRNAs of equine arteritis virus contain a common leader sequence.

During the replication of equine arteritis virus (EAV) six subgenomic mRNAs are synthesized. We present evidence that the viral mRNAs form a 3'-coterminal nested set and contain a common leader sequence of 208 nucleotides which is encoded by the 5'-end of the genome. The leader is joined to the bodies of mRNA 5 and 6 at positions defined by the sequence 5' UCAAC 3'. The part of the leader sequence flanking the UCAAC motif is very similar to the 5'-splice site of the Tetrahymena pre-rRNA. A possible internal guide sequence has been identified 43 nucleotides downstream of the leader sequence on the genome. Hybridization analysis shows that all EAV intracellular RNAs contain the leader sequence. These data imply that the viral subgenomic mRNAs are composed of leader and body sequences which are non-contiguous on the genome.

Animals↗
PubMed1991

Casein kinase II from Caenorhabditis elegans. Cloning, characterization, and developmental regulation of the gene encoding the beta subunit.

Complementary DNAs encoding the beta subunit of casein kinase II (CKII beta) from the nematode Caenorhabditis elegans were cloned and sequenced. The predicted beta subunit polypeptide comprises 234 amino acid residues and has a Mr of 26,452. CKII beta is not homologous with other types of proteins. In synchronously developing C. elegans the abundance of the 1.3-kilobase mRNA for CKII beta varies in parallel with the level of mRNA encoding the catalytic subunit (alpha) of CKII. Thus, the developmental expression of CKII subunits is controlled coordinately and pretranslationally. CKII beta and CKII alpha mRNAs are enriched 5-10-fold in C. elegans embryos relative to their concentrations at several other stages of nematode development. A 3.8-kilobase pair segment of C. elegans DNA that contains the CKII beta gene and an extensive 5'-flanking region was cloned and sequenced. The CKII beta gene is divided into 6 exons by introns ranging from 49 to 533 base pairs in length. The first exon encodes 88 nucleotides of 5'-untranslated mRNA. Exon 2 (72 base pairs) contains the initiator Met codon and only 5 additional codons. Exons 3-6 encode 52, 63, 64, and 49 amino acid residues, respectively. The 5' terminus of CKII beta mRNA is modified post-transcriptionally by trans-splicing with a leader sequence of 22 nucleotides. The CKII beta gene was mapped to a position on C. elegans chromosome 2 that is in close proximity to the lin-11 gene.

Amino Acid Sequence↗
PubMed1987

In vitro synthesis of vaccinia virus late mRNA containing a 5' poly(A) leader sequence.

We have shown that an extract made from HeLa cells harvested 6 hr after infection with vaccinia virus can transcribe a duplex DNA template containing a late viral gene. S1 nuclease analyses using genomic and synthetic probes indicated that the 5' ends of RNA synthesized in vitro are similar to those of RNA made in vivo and contain 5' poly(A) sequences contiguous with the translation initiation codon. Kinetic analysis of RNA synthesized in vitro demonstrated that a correctly initiated and 5' polyadenylylated product appeared within 5 min after transcription reactions were started. A cis-splicing mechanism of poly(A) addition can be ruled out because the DNA template used in vitro had no poly(dT) sequence and could contain as few as 37 base pairs upstream of the start of the RNA. In addition, we found that a point mutation in the first of two consecutively encoded adenylate residues preceding the ATG initiation codon abolished transcription in vitro. These data are consistent with at least three models: (i) RNA polymerase initiates RNA synthesis with a run of adenylate residues; (ii) a poly(A) primer is used for initiation; or (iii) the poly(A) leader is rapidly and efficiently attached to the RNA by ligation.

Cell-Free System↗
PubMed1997

cut-1-like genes of Ascaris lumbricoides.

Three genomic fragments homologous to cut-1 of Caenorhabditis elegans (C. elegans) have been identified in the intestinal parasitic nematode Ascaris lumbricoides (A. lumbricoides). Two of these fragments identify one region of the A. lumbricoides genome; they are separated by 8-9 kb and have opposite orientation, with the direction of transcription converging toward the center of the region. The third gene, which has been studied more completely, is in a different region of the genome separated from the first one by not less than 12-15 kb. The complete genomic sequence of this third gene has been determined. cDNA overlapping clones were obtained from adult A. lumbricoides RNA via the rapid amplification of cDNA ends (RACE) procedure [Frohman et al., 1988. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998-9002] and sequenced. The mature mRNA of this gene, which we have named ascut-1, is trans-spliced to the spliced leader sequence of nematodes (SL1) [Krause, M., Hirsh, D., 1987. A trans-spliced leader sequence on actin mRNA in C. elegans. Cell 49, 753-761]. The mRNA is 1684 nt long plus the poly(A) tail and contains four exons with a 138 nt untranslated 5' leader and a 388 nt untranslated 3' tail. Conceptual translation of the coding sequence shows a protein of 385 aa with a signal peptide of 16 aa. The protein shows very high homology with CECUT-1, the product of the C. elegans gene cut-1 and with other cuticlin proteins of nematodes. A 262 amino acids region which is strongly conserved between these proteins seems to identify a group of proteins, so far restricted to nematodes, for which the name CUT-1-like is proposed.

Amino Acid Sequence↗
PubMed1989

Disruption of the yeast nuclear PET54 gene blocks excision of mitochondrial intron aI5 beta from pre-mRNA for cytochrome c oxidase subunit I.

The nuclear PET54 gene of Saccharomyces cerevisiae was cloned and a pet54::LEU2 gene disruption strain was constructed. Analysis of the phenotype of this strain revealed a defect in expression of two mitochondrial genes: COX1, which encodes cytochrome c oxidase subunit I, and COX3, which encodes cytochrome c oxidase subunit III. The defect in COX1 gene expression in the pet54 mutant was shown to be the result of inefficient excision of COX1 intron aI5 beta. Two lines of evidence indicate that inefficient excision of intron aI5 beta is the sole defect in COX1 gene expression. First, a pet54::LEU2 cytoductant bearing the 'short' mitochondrial genome that lacks both COX1 introns aI5 alpha and aI5 beta is defective only in COX3 gene expression and not in COX1 mRNA splicing or mRNA translation. Second, Northern analysis of COX1 transcipts from the pet54 mutant showed that a 3.8 kb COX1 transcript containing unexcised intron aI5 beta and lacking intron aI5 alpha is accumulated while the amount of 2.2 kb mature COX1 mRNA is diminished. In an effort to relate the role of the PET54 gene product in splicing of COX1 pre-mRNA to the previously characterized role for PET54 in translation of mitochondrial COX3 mRNA, the sequence of the PET54-responsive portion of the COX3 5' untranslated leader region was compared to the COX1 intron aI5 beta sequence. Two blocks of RNA sequence present in COX3 have similar counterparts within intron aI5 beta of COX1. The possibility that the PET54 protein binds to one or the other of these blocks of RNA sequence and the potential consequences of this interaction are discussed.

Base Sequence↗
PubMed1995

Caenorhabditis elegans UBC-1, a ubiquitin-conjugating enzyme homologous to yeast RAD6/UBC2, contains a novel carboxy-terminal extension that is conserved in nematodes.

The RAD6/UBC2 gene from Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme involved in DNA repair, induced mutagenesis, and sporulation. Here we report the isolation and characterization of the Caenorhabditis elegans RAD6 homolog designated ubc-1. Ubc-1 encodes a 21.5-kD protein that shares considerable identity with RAD6 (66%) as well as with other RAD6 homologs, including Schizosaccharomyces pombe rhp6+ (70%), Drosophila melanogaster Dhr6 (83%), and the two human homologs HHR6A and HHR6B (84% and 83%, respectively). However, UBC-1 is distinct in being the only known RAD6 homolog, other than RAD6 itself, with a carboxy-terminal extension. Analysis of UBC-1 homologs from C. briggsae and Ascaris suum show that the presence of the carboxy-terminal extension is conserved in nematodes. When constitutively expressed from the yeast promoter ADH1, ubc-1 complements the DNA repair functions in a S. cerevisiae rad6 delta mutant. Surprisingly, ubc-1 fails to complement the sporulation function of RAD6, despite its possession of an acidic carboxy-terminal tail. C. elegans UBC-1 is capable of forming a thiol-ester bond with ubiquitin, but, unlike RAD6, is unable to transfer ubiquitin to histone H2B in vitro. Both cis and trans splicing are involved in the maturation of the ubc-1 transcript. The presence of the SL2 trans-splice leader in the ubc-1 transcript suggests that ubc-1 may be co-transcribed as part of a polycistronic message.

Amino Acid Sequence↗
PubMed1993

Characterization of cDNA clones for the 2-methyl branched-chain enoyl-CoA reductase. An enzyme involved in branched-chain fatty acid synthesis in anaerobic mitochondria of the parasitic nematode Ascaris suum.

The 2-methyl branched-chain enoyl-CoA reductase plays a pivotal role in the reversal of beta-oxidation operating in anaerobic mitochondria of the parasitic nematode Ascaris suum. An affinity-purified polyclonal anti-serum against the reductase was used to screen a cDNA library constructed in lambda gt11 with poly(A)+ RNA from adult A. suum muscle. A 1.2-kilobase partial cDNA clone was isolated, subcloned, and sequenced in both directions. Additional sequence at the 5' end of the mRNA was determined by the RACE (rapid amplification of cDNA ends) procedure. Nucleotide sequence analysis of the cDNAs revealed the 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, an open reading frame of 1236 nucleotides and a 3'-untranslated sequence of 109 nucleotides including a short poly(A) tail 14 nucleotides from a polyadenylation signal (AATAAA). The open reading frame encoded a 396-amino acid sequence (M(r) 43,046) including a 16-amino acid leader peptide. Two-dimensional gel electrophoresis of the purified reductase yielded multiple spots with two distinct but overlapping amino-terminal amino acid sequences. Both sequences overlapped with the sequence predicted from the mRNA, and one of the sequences was identical to the predicted sequence. Comparison of the ascarid sequence with that of mammalian acyl-CoA dehydrogenases revealed a high degree of sequence identity, suggesting that these enzymes may have evolved from a common ancestral gene even though the ascarid enzyme functions as a reductase, not as a dehydrogenase. Immunoblotting of A. suum larval stages and adult tissues with antisera that cross-reacted with each of the spots separated on two-dimensional gels suggested that the reductase was only found in adult muscle. Northern blotting using the partial cDNA revealed a hybridization band of about 1.5 kilobases and also suggested that the enzyme was tissue-specific and developmentally regulated in agreement with the results of the immunoblotting.

Amino Acid Sequence↗
PubMed1989

Mapping the termini and intron of the spliced immediate-early transcript of equine herpesvirus 1.

Equine herpesvirus 1 (EHV-1) has been shown to synthesize a 6.0-kilobase (kb) species of immediate-early (IE) mRNA in productively infected cells. This IE gene region maps within the outer portion (map units 0.79 to 0.83 and 0.96 to 1.00) of the two inverted repeat segments of the short genomic region, and elucidation of its DNA sequence has revealed multiple potential open reading frames (ORFs), including a major ORF of 4,461 nucleotides (F. J. Grundy, R. P. Baumann, and D. J. O'Callaghan, Virology 172:223-236, 1989). Analyses of IE polypeptides synthesized in EHV-1-infected cells (in vivo) and in vitro translation of hybrid-selected IE mRNA indicated that multiple species of IE proteins are encoded by this IE mRNA species. To address the nature of the 6.0-kb IE RNA species, Northern (RNA) blot hybridization, S1 nuclease mapping, and primer extension analyses have been employed. These data revealed that no major introns were detected within the body of the IE transcript. However, the IE mRNA was shown to be spliced at the 5' terminus, such that a 372-base intron containing two small ORFs (19 and 51 amino acids) was removed from the leader region of the transcript. This splicing event reduced the leader region from 625 to 253 bases. S1 and primer extension analyses of the 5' terminus of this transcript revealed that the transcription initiation site is located 24 to 26 bases downstream of the consensus TATAAA motif. The 3' transcription termination site was mapped by S1 nuclease analysis to approximately 10 to 20 bases downstream of the polyadenylation signal, AATAAA. The distance from the stop codon of the major ORF to the polyadenylation site is approximately 300 bases. Results from S1 nuclease experiments indicated that splicing does not occur at the 3' terminus. These studies indicated that the EHV-1 6.0-kb IE mRNA is spliced at the 5' terminus and that alternative splicing of this transcript may function in regulating translation of the IE mRNA species.

Animals↗
PubMed2000

5'-heterogeneity of glucocorticoid receptor messenger RNA is tissue specific: differential regulation of variant transcripts by early-life events.

Glucocorticoid receptor (GR) gene expression is regulated in a complex tissue-specific manner, notably by early-life environmental events that program tissue GR levels. We have identified and characterized several new rat GR mRNAs. All encode a common protein, but differ in their 5'-leader sequences as a consequence of alternate splicing of, potentially, 11 different exon 1 sequences. Most are located in a 3-kb CpG island, upstream of exon 2, that exhibits substantial promoter activity in transfected cells. Ribonuclease (RNase) protection analysis demonstrated significant levels of six alternate exons 1 in vivo in rat, with differences between liver, hippocampus, and thymus reflecting tissue-specific differences in promoter activity. Two of the alternate exons 1 (exons 1(6) and 1(10)) were expressed in all tissues examined, together present in 77-87% of total GR mRNA. The remaining GR transcripts contained tissue-specific alternate first exons. Importantly, tissue-specific first exon usage was altered by perinatal environmental manipulations. Postnatal handling, which permanently increases GR in the hippocampus, causing attenuation of stress responses, selectively elevated GR mRNA containing the hippocampus-specific exon 1(7). Prenatal glucocorticoid exposure, which increases hepatic GR expression and produces adult hyperglycemia, decreased the proportion of hepatic GR mRNA containing the predominant exon 1(10), suggesting an increase in a minor exon 1 variant. Such tissue specificity of promoter usage allows differential GR regulation and programming.

Alternative Splicing↗
PubMed1995

Differentiation-dependent transcription of the epidermodysplasia verruciformis-associated human papillomavirus type 5 in benign lesions.

Human papillomavirus type 5 (HPV 5) induces cutaneous lesions and persists in skin carcinomas of patients with epidermodysplasia verruciformis (EV). We investigated the expression pattern of HPV 5 in biopsies from benign skin lesions of EV patients by cDNA analysis and in situ hybridization. Nine different cDNAs could be generated from total RNA of one of these lesions by reverse transcription and PCR amplification with HPV 5-specific primers. We could identify two major splice donors: one was found in the E6-proximal part of the noncoding region (NCR), and the other just downstream of the first ATG codon of ORF E1. Each of the characterized transcripts was processed at one or the other donor site and the two corresponding leader exons were found in combination with both 3'-early and late exons. Two transcripts appear to be specific for EV-associated papillomaviruses: one species might encode an E1--E2C fusion protein, and the other mRNA (NCR/E2) is probably encoding for the full-length E2 protein. According to the results of the cDNA analysis, riboprobes were designed for in situ hybridization experiments to study the cell differentiation-dependent expression of the different exons. Only the E7/E1 and E4 probes led to strong signals almost throughout the epithelium. The signals generated by the 5'-E2 and E1 probe increased with cell differentiation and were mainly confined to the nucleus. The NCR, E6, E7, L2, and L1 probes yielded more or less strong signals in the terminally differentiated epidermal layers. The difference in the cell differentiation-dependent expression of the 5'-early region exon (probe E7/1) and L2/L1 exons may point to a differentiation-dependent processing of transcripts.

Base Sequence↗
PubMed1996

Genes expressed in Brugia malayi infective third stage larvae.

We have used a tag sequencing approach to survey genes expressed in the third stage infective larvae of the human filarial nematode parasite Brugia malayi. RNA was isolated from late vector-stage L3 larvae after days 9 or 10 of infection in mosquitos, and converted to cDNA by reverse transcriptase. Double-stranded cDNA was produced by either conventional methods (non-SL cDNA library) or by PCR using the nematode spliced leader (SLI) and oligo(dT) primers (SL cDNA library). Two clone libraries (one from SL and one from non-SL cDNAs) were constructed in lambda ZapII. A set of these full-length clones was selected and 596 inserts were sequenced from the 5' end. We have identified 364 B. malayi genes (the majority of which are new) that encode housekeeping proteins, structural proteins, proteins of immediate immunological or drug-discovery interest as well as a large class of novel sequences which may prove to have significant involvement in host invasion. Extensive, genome-wide approaches to the analysis of larval gene expression are now possible for B. malayi. We present several examples of this approach.

Amino Acid Sequence↗
PubMed1987

Varicella-zoster virus as a live vector for the expression of foreign genes.

The previous demonstration of the efficacy and tolerability of the Oka strain of varicella-zoster virus (VZV) in clinical trials involving vaccination of both normal and immunocompromised individuals has laid the foundation for its use in preventing chickenpox. In this context, VZV could be useful as a vector for vaccinating against other infectious agents as well. As an initial application, a live recombinant VZV expressing Epstein-Barr virus (EBV) membrane glycoproteins (gp350/220) was generated by inserting a gene fusion of the VZV gpI promoter and hydrophobic leader-encoding sequence with the gp350/220 coding sequence into the thymidine kinase (TK) gene of VZV (Oka). Insertion of the foreign DNA into the thymidine kinase gene was demonstrated by Southern blot analysis and the ability of the recombinant virus to replicate in the presence of bromodeoxyuridine. RNA splicing, glycosylation, and plasma membrane presentation of gp350/220 in cells infected with the recombinant virus were similar to those seen in EBV-infected cells. In addition, the expression of VZV-specific glycoproteins was unaltered by the concomitant expression of this large foreign glycoprotein. Thus, VZV can be used as a live viral vector for active immunization against EBV and other pathogens.

Cell Line↗
PubMed1991

U small nuclear ribonucleoprotein requirements for nematode cis- and trans-splicing in vitro.

In nematodes, a fraction of mRNAs acquires a common 22-nucleotide 5'-terminal spliced leader sequence via a trans-splicing reaction. The same premessenger RNAs which receive the spliced leader are also processed by conventional cis-splicing. Whole cell extracts prepared from synchronous embryos of the parasitic nematode Ascaris lumbricoides catalyze both cis- and trans-splicing. We have used this cell-free system and oligodeoxynucleotide directed RNase H digestion to assess the U small nuclear RNA requirements for nematode cis- and trans-splicing. These experiments indicated that both cis- and trans-splicing require intact U2 and U4/U6 small nuclear ribonucleoproteins (snRNPs). However, whereas cis-splicing displays the expected requirement for an intact U1 snRNP, trans-splicing is unaffected when approximately 90% of U1 snRNP is degraded. These results suggest that 5' splice site identification differs in nematode cis- and trans-splicing.

Animals↗
PubMed1979

SV40 recombinants carrying rabbit beta-globin gene coding sequences.

We have constructed and propagated two SV40 recombinants in which different portions of the viral late gene region are replaced by a rabbit beta-globin coding sequence derived from a cloned cDNA (Maniatis et al., 1976). One of these recombinants, which retains the promoter, leader and intervening sequences for a viral late mRNA, directs the synthesis of a stable SV40-globin hybrid transcript. Using a sensitive radioimmunoassay, we have shown that this globin RNA is translated in infected monkey cells. A second recombinant, which retains the late region promoter but lacks the RNA splicing region, produces neither a stable globin transcript nor any detectable beta-globin. These experiments indicate that some sequence within a 500 bp SV40 segment, presumably the splicing region, is required for the accumulation of stable mRNA. They also demonstrate how hybrid transducing viruses can be used both to characterize viral regulatory sequences and to produce new gene products in cultured cells.

Animals↗