Sequence of the nucleocapsid gene from murine coronavirus MHV-A59.

The nucleotide sequence of the RNA encoding the nucleocapsid protein of coronavirus MHV-A59 has been determined. Copy DNA was prepared from mRNA isolated from virally infected cells, fragmented and cloned in the phage vector M13 mp8 for direct sequence determination. A sequence of 1817 nucleotides, adjacent to the viral poly-A tail, was obtained. It contains a single long open reading frame encoding a protein of mol. wt. 49660, which is enriched in basic residues.     

Laminated cisternae of the rough endoplasmic reticulum induced by coronavirus MHV-A59 infection

The infection of murine fibroblasts of the sac- line with a coronavirus, mouse hepatitis virus strain A59 (MHV-A59), results in a novel modification to some cisternae of the rough endoplasmic reticulum (RER). From 8 hours post infection (h.p.i.) we see in thin sections pairs of cisternae closely, stably and uniformly aligned. Serial sectioning shows that the regions of pairing or lamination extend for many thousands of nm in two dimensions, with the spacing between the juxtaposed membranes remaining very uniform at about 18 nm. These structures appear coincident with the onset of accumulation of the viral glycoprotein E1 in the RER membrane but 2 hours after the viral glycoprotein E2 can first be detected there. Ribosomes are excluded from the paired cisternal surfaces, while budding of progeny virions has never been seen at the cisternal membranes facing the cytosol, although ribosomes bind there. The lumina of paired cixternae are usually devoid of virions which, however, accumulate in areas where the paired cisternae diverge. Electron immunocytochemistry shows that both E1 and E2 glycoproteins are abundant in the paired cisternae. Following labelling for the E1 glycoprotein we see a periodic fine structure, rows of "beads" with a centre to centre spacing of about 7.5 nm, in the region between the paired membranes. In oblique sections of this region in cells fixed as if for the immunoperoxidase labelling, but omitting all its steps we see parallel rows of "beads" separated by about 7 nm. We suggest that the membrane spanning viral glycoprotein E1 together with viral nucleocapsids may be involved in laminating cisternae of the RER.     

Mapping of Drosophila mutations using site-specific male recombination.

Although recombination does not usually occur in the male Drosophila germline, site-specific recombination can be induced at the ends of P elements. This finding suggested that male recombination could be used to map Drosophila mutations. In this article, we describe the general method and its application to the mapping of two EMS-induced female-sterile mutations, grauzone and cortex. Within two months, the grauzone gene was mapped relative to seven different P-element insertion sites, and cortex was mapped relative to 23 different P-elements. The results allowed us to map grauzone to a region of about 50 kb, and cortex distal to the chromosomal region 33E. These experiments demonstrate that P-element-induced site-specific male recombination is an efficient and general method to map Drosophila autosomal mutations.     

Homologous terminal sequences of the genome double-stranded RNAs of bluetongue virus.

The 3'-terminal sequences of the 10 double-stranded RNA genome segments of bluetongue virus (serotypes 10 and 11) were determined. The double-stranded RNAs were 3' labeled with [5'-32P]pCp and resolved into 10 segments by electrophoresis. After denaturation, the two complementary strands of segments 4 through 10 were resolved into fast- and slow-migrating species by polyacrylamide gel electrophoresis, and their 3' end sequences were determined. Complete RNase T1 digestion of the individual 3'-labeled double-stranded RNA segments yielded two labeled oligonucleotides, one of which migrated faster than the other on 20% polyacrylamide-7 M urea gels. Sequence analyses of the two oligonucleotides of segments 4 through 10 confirmed the corresponding RNA sequence data. For RNA segments 1 through 3 the oligonucleotide analyses gave comparable results. The 3'-terminal sequences of the fast-migrating RNA species were HOCAAUUU. . . ; those of the slow-migrating RNA species were HOCAUUCACA. . . . Similar results were obtained for double-stranded RNA from bluetongue virus serotypes 10 and 11. Beyond the common termini, the sequences for each segment varied considerably.     

An RNA ligase-mediated method for the efficient creation of large, synthetic RNAs

RNA ligation has been a powerful tool for incorporation of cross-linkers and nonnatural nucleotides into internal positions of RNA molecules. The most widely used method for template-directed RNA ligation uses DNA ligase and a DNA splint. While this method has been used successfully for many years, it suffers from a number of drawbacks, principally, slow and inefficient product formation and slow product release, resulting in a requirement for large quantities of enzyme. We describe an alternative technique catalyzed by T4 RNA ligase instead of DNA ligase. Using a splint design that allows the ligation junction to mimic the natural substrate of RNA ligase, we demonstrate several ligation reactions that appear to go nearly to completion. Furthermore, the reactions generally go to completion within 30 min. We present data evaluating the relative importance of various parameters in this reaction. Finally, we show the utility of this method by generating a 128-nucleotide pre-mRNA from three synthetic oligoribonucleotides. The ability to ligate synthetic or in vitro transcribed RNA with high efficiency has the potential to open up areas of RNA biology to new functional and biophysical investigation. In particular, we anticipate that site-specific incorporation of fluorescent dyes into large RNA molecules will yield a wealth of new information on RNA structure and function.     

Homologous recombination between overlapping thymidine kinase gene fragments stably inserted into a mouse cell genome.

We have constructed a substrate to study homologous recombination between adjacent segments of chromosomal DNA. This substrate, designated lambda tk2 , consists of one completely defective and one partially defective herpes simplex virus thymidine kinase (tk) gene cloned in bacteriophage lambda DNA. The two genes have homologous 984-base-pair sequences and are separated by 3 kilobases of largely vector DNA. When lambda tk2 DNA was transferred into mouse LMtk- cells by the calcium phosphate method, rare TK+ transformants were obtained that contained many (greater than 40) copies of the unrecombined DNA. Tk- revertants, which had lost most of the copies of unrecombined DNA, were isolated from these TK+-transformed lines. Two of these Tk- lines were further studied by analysis of their reversion back to the Tk+ phenotype. They generated ca. 200 Tk+ revertants per 10(8) cells after growth in nonselecting medium for 5 days. All of these Tk+ revertants have an intact tk gene reconstructed by homologous recombination; they also retain various amounts of unrecombined lambda tk2 DNA. Southern blot analysis suggested that at least some of the recombination events involve unequal sister chromatid exchanges. We also tested three agents, mitomycin C, 12-O-tetradecanoyl-phorbol-13-acetate, and mezerein, that are thought to stimulate recombination to determine whether they affect the reversion from Tk- to Tk+. Only mitomycin C increased the number of Tk+ revertants.     

Repair and mutagenesis of the genome of a deletion mutant of the coronavirus mouse hepatitis virus by targeted RNA recombination.

The genetic characterization of a nucleocapsid (N) protein mutant of the coronavirus mouse hepatitis virus (MHV) is described. The mutant, Albany 4 (Alb4), is both temperature sensitive and thermolabile. Analysis of the progeny of a mixed infection showed that the defective Alb4 allele is recessive to wild type, and its gene product is diffusible. The N protein of Alb4 was found to be smaller than its wild-type counterpart, and sequence analysis of the Alb4 N gene revealed that it contains an internal deletion of 87 nucleotides, producing an in-frame deletion of 29 amino acids. All of these properties of Alb4 made it ideal for use as a recipient in a targeted RNA recombination experiment in which the deletion in Alb4 was repaired by recombination with synthetic RNA7, the smallest MHV subgenomic mRNA. Progeny from a cotransfection of Alb4 genomic RNA and synthetic RNA7 were selected for thermal stability. Polymerase chain reaction analysis of candidate recombinants showed that they had regained the material that is deleted in the Alb4 mutant. They also had acquired a five-nucleotide insertion in the 3' untranslated region, which had been incorporated into the synthetic RNA7 as a molecular tag. The presence of the tag was directly verified, as well, by sequencing the genomic RNA of purified recombinant viruses. This provided a clear genetic proof that the Alb4 phenotype was due to the observed deletion in the N gene. In addition, these results demonstrated that it is possible to obtain stable, independently replicating progeny from recombination between coronavirus genomic RNA and a tailored, synthetic RNA species.     

Replication of coronavirus MHV-A59 in sac- cells: determination of the first site of budding of progeny virions

During infection of sac- cells by murine coronavirus MHV A59 the intracellular sites at which progeny virions bud correlate with the distribution of the viral glycoprotein E1. Budding is first detectable by electron microscopy at 6 to 7 hours post infection in small, smooth, perinuclear vesicles and tubules in a region transitional between the rough endoplasmic reticulum and the Golgi apparatus. At later times the rough endoplasmic reticulum becomes the major site of budding and accumulation of progeny virus particles. Indirect immunofluorescence microscopy shows that E1 is confined at 6 hours post infection to the perinuclear region while at later times it also accumulates in the endoplasmic reticulum. At 6 hours post infection the second viral glycoprotein, E2, is distributed throughout the endoplasmic reticulum and is not restricted to the site at which budding begins. Core protein, the third protein in virions, can be detected 2 hours before E1 is detectable and budding begins, and at 6 hours post infection it is distributed throughout the cytosol. We conclude that the time and the site at which the maturation of progeny virions occurs is determined by the accumulation of glycoprotein E1 in intracellular membranes. Only rarely do progeny virions bud directly into the cisternae of the Golgi apparatus but at least some already budded virions are transported to the Golgi apparatus where they occur in structures some of which also contain TPPase, a trans Golgi marker.     

A cell-free recombination system for site-specific integration of multigenic shuttle plasmids into the herpes simplex virus type 1 genome.

This report describes a novel method for complementation studies of defective herpes simplex virus (HSV) genes. Viral test gene and nonviral reporter gene cassettes were rapidly integrated into the HSV genome in a site-specific and reversible manner by using the P1 phage-based Cre-lox recombination system. Shuttle plasmids contained a functional loxP recombination site, an expressible form of the bacterial lacZ gene, and a copy of the wild-type glycoprotein B (gB) gene or double mutant gB allele containing both a temperature-sensitive (ts) mutation and a syncytium (syn)-forming mutation. A recipient viral genome, K delta T::lox1, was constructed from the HSV type 1 (syn) gB-deficient mutant virus, K delta T, by marker transfer of the loxP recombination site into the viral thymidine kinase locus. Shuttle plasmids of up to 12.9 kb in length were recombined with high efficiency (11 to 20%) into the K delta T::lox1 genome in cell-free, Cre-mediated recombination reactions. Expression of a functional wild-type or double mutant gB polypeptide complemented the nonfunctional polypeptide expressed from the deleted, normal gB locus and allowed production of either wild-type or Syn- plaques on Vero cells. The latter recombinant virus was also ts for growth. The ability to express viral genes from plasmids which can be shuttled into and out of the HSV genome in cell-free recombination reactions makes this a powerful method for performing genetic studies of the biologic properties of viral gene products.     

The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome

We have transferred the site-specific recombination system of the yeast 2 micron plasmid, the FLP recombinase and its recombination targets (FRTs), into the genome of Drosophila. Flies were transformed with an FLP gene under the control of hsp70 regulatory sequences and with a white gene flanked by FRTs. The heat-induced recombinase catalyzes recombination between FRTs, causing loss of white (seen somatically as white patches in the eye) and, less frequently, gain of white (seen as dark-red patches). Loss and gain frequencies vary with the severity of the heat shock, and patterns of mosaicism vary with the developmental stage at which the heat shock is applied. The recombinase is also active in the germline, producing white-eyed and dark-red-eyed progeny.     

Homologous recombination in plant cells is enhanced by in vivo induction of double strand breaks into DNA by a site-specific endonuclease.

Induction of double strand breaks (DSBs) is coupled to meiotic and mitotic recombination in yeast. We show that also in a higher eukaryote induction of DSBs is directly correlated with a strong enhancement of recombination frequencies. We cotransfected Nicotiana plumbaginifolia protoplasts with a plasmid carrying a synthetic I-SceI gene, coding for a highly sequence specific endonuclease, together with recombination substrates carrying an I-SceI-site adjacent to their homologous sequences. We measured efficiencies of extrachromosomal recombination, using a well established transient beta-glucuronidase (GUS) assay. GUS enzyme activities were strongly increased when a plasmid carrying the I-SceI gene in sense but not in antisense orientation with respect to the promoter was included in the transfections. The in vivo induced DSBs were detected in the recombination substrates by Southern blotting, demonstrating that the yeast enzyme is functional in plant cells. At high ratios of transfected I-SceI-genes to I-SceI-sites the majority of the I-SceI-sites in the recombination substrates are cleaved, indicating that the induction of the DSBs is the rate limiting step in the described recombination reaction. These results imply that in vivo induction of transient breaks at specific sites in the plant genome could allow foreign DNA to be targeted to these sites via homologous recombination.     

Biochemical evidence of recombination within the unsegmented RNA genome of aphthovirus.

Four different pairs of temperature-sensitive mutants, derived from the same strain of aphthovirus, were crossed by using an infectious center recombination test. Each parental mutant carried an unselected marker affecting the isoelectric point of a virus-coded polypeptide; progeny of the crosses, able to grow at the nonpermissive temperature, were screened for these unselected markers. Polypeptide charge was shown to be a stable, inheritable character, and three of the four crosses yielded genetic recombinants that possessed the polypeptide marker from both parents. Peptide fingerprinting and high-resolution isoelectric focusing of the polypeptides ruled out the explantation that these viruses were generated by mutation.     

Conditionally replicating plasmid vectors that can integrate into the Klebsiella pneumoniae chromosome via bacteriophage P4 site-specific recombination.

P4 is a satellite phage of P2 and is dependent on phage P2 gene products for virion assembly and cell lysis. Previously, we showed that a virulent mutant of phage P4 (P4 vir1) could be used as a multicopy, autonomously replicating plasmid vector in Escherichia coli and Klebsiella pneumoniae in the absence of the P2 helper. In addition to establishing lysogeny as a self-replicating plasmid, it has been shown that P4 can also lysogenize E. coli via site-specific integration into the host chromosome. In this study, we show that P4 also integrates into the K. pneumoniae chromosome at a specific site. In contrast to that in E. coli, however, site-specific integration in K. pneumoniae does not require the int gene of P4. We utilized the alternative modes of P4 lysogenization (plasmid replication or integration) to construct cloning vectors derived from P4 vir1 that could exist in either lysogenic mode, depending on the host strain used. These vectors carry an amber mutation in the DNA primase gene alpha, which blocks DNA replication in an Su- host and allows the selection of lysogenic strains with integrated prophages. In contrast, these vectors can be propagated as plasmids in an Su+ host where replication is allowed. To demonstrate the utility of this type of vector, we show that certain nitrogen fixation (nif) genes of K. pneumoniae, which otherwise inhibit nif gene expression when present on multicopy plasmids, do not exhibit inhibitory effects when introduced as merodiploids via P4 site-specific integration.     

Highly efficient generation of recombinant baculoviruses by enzymatically medicated site-specific in vitro recombination.

We have used the Cre-lox system of bacteriophage P1 to develop a highly efficient in vitrosystem for construction of recombinant baculoviruses. A positive visual selection has been included to make identification of recombinant viral progeny rapid and straightforward. We report recombination frequencies as high as 5 x 10(7) recombinants/micrograms starting plasmid DNA and under certain conditions, up to 50% of the viral progeny are recombinants. Genes inserted into the baculovirus genome can be readily recovered in a simple one step process and re-inserted after manipulation if required. We have confirmed the structure of recovered plasmids by diagnostic restriction endonuclease digestion and the structure of recombinant viral genomes by Southern analysis. Possible uses and the significance of the system are discussed and experiments currently being done to improve it are described.     

The primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus MHV-A59; a highly conserved polymerase is expressed by an efficient ribosomal frameshifting mechanism.

Sequence analysis of a substantial part of the polymerase gene of the murine coronavirus MHV-A59 revealed the 3' end of an open reading frame (ORF1a) overlapping with a large ORF (ORF1b; 2733 amino acids) which covers the 3' half of the polymerase gene. The expression of ORF1b occurs by a ribosomal frameshifting mechanism since the ORF1a/ORF1b overlapping nucleotide sequence is capable of inducing ribosomal frameshifting in vitro as well as in vivo. A stem-loop structure and a pseudoknot are predicted in the nucleotide sequence involved in ribosomal frameshifting. Comparison of the predicted amino acid sequence of MHV ORF1b with the amino acid sequence deduced from the corresponding gene of the avian coronavirus IBV demonstrated that in contrast to the other viral genes this ORF is extremely conserved. Detailed analysis of the predicted amino acid sequence revealed sequence elements which are conserved in many DNA and RNA polymerases.     

Influenza B viruses with site-specific mutations introduced into the HA gene.

We have succeeded in engineering changes into the genome of influenza B virus. First, model RNAs containing the chloramphenicol acetyltransferase gene flanked by the noncoding sequences of the HA or NS genes of influenza B virus were transfected into cells which were previously infected with an influenza B helper virus. Like those of the influenza A viruses, the termini of influenza B virus genes contain cis-acting signals which are sufficient to direct replication, expression, and packaging of the RNA. Next, a full-length copy of the HA gene from influenza B/Maryland/59 virus was cloned. Following transfection of this RNA, we rescued transfectant influenza B viruses which contain a point mutation introduced into the original cDNA. A series of mutants which bear deletions or changes in the 5' noncoding region of the influenza B/Maryland/59 virus HA gene were constructed. We were able to rescue viruses which contained deletions of 10 or 33 nucleotides at the 5' noncoding region of the HA gene. The viability of these viruses implies that this region of the genome is flexible in sequence and length.     

Homologous recombination mediated by two palindromic repeated sequences in the mitochondrial genome of Oryza

Palindromic repeated sequences (PRSs) are distributed in at least ten regions of the mitochondrial (mt) genome of rice and are, apparently, mobile. In the present study, we examined the possibility of homologous recombination via some PRSs during the course of evolution of Oryza. We first performed Southern hybridization of the DNA from 11 species (18 strains) of Oryza in order to identify the distribution of PRSs in the mitochondrial genome of Oryza. The hybridization patterns revealed genome type-specific and/or species-specific variations. We speculated that homologous recombination via some PRSs might have made a contribution to such variations. After subsequent polymerase chain reaction, Southern hybridization and sequencing, we concluded that homologous recombination mediated by two PRSs occurred in the mtDNA of Oryza after divergence of the BB genome type and the other genome types of Oryza. Evidence was obtained that some PRSs were involved in both insertion and recombination events during the evolution of Oryza. Our results indicate, therefore, that PRSs have contributed considerably to the polymorphism of Oryza mtDNAs.     

Homologous RNA recombination in brome mosaic virus: AU-rich sequences decrease the accuracy of crossovers.

Brome mosaic virus, a tripartite positive-stranded RNA virus of plants, was used for the determination of sequence requirements of imprecise (aberrant) homologous recombination. A 23-nucleotide (nt) region that included a 6-nt UUAAAA sequence (designated the AU sequence) common between wild-type RNA2 and mutant RNA3 supported both precise and imprecise homologous recombination, though the latter occurred with lower frequency. Doubling the length of the 6-nt AU sequence in RNA3 increased the incidence of imprecise crossovers by nearly threefold. Duplication or triplication of the length of the AU sequence in both RNA2 and RNA3 further raised the frequency of imprecise crossovers. The majority of imprecise crosses were located within or close to the extended AU sequence. Imprecise recombinants contained either nucleotide substitutions, nontemplated nucleotides, small deletions, or small sequence duplications within the region of crossovers. Deletion of the AU sequence from the homologous region in RNA3 resulted in the accumulation of only precise homologous recombinants. Our results provide experimental evidence that AU sequences can facilitate the formation of imprecise homologous recombinants. The generation of small additions or deletions can be explained by a misannealing mechanism within the AU sequences, while replicase errors during RNA copying might explain the occurrence of nucleotide substitutions or nontemplated nucleotides.     

Cre/ lox site-specific recombination controls the excision of a transgene from the rice genome

The Cre/lox site-specific recombination controls the excision of a target DNA segment by recombination between two lox sites flanking it, mediated by the Cre recombinase. We have studied the functional expression of the Cre/lox system to excise a transgene from the rice genome. We developed transgenic plants carrying the target gene, hygromycin phosphotransferase (hpt) flanked by two lox sites and transgenic plants harboring the Cre gene. Each lox plant was crossed with each Cre plant reciprocally. In the Cre/lox hybrid plants, the Cre recombinase mediates recombination between two lox sites, resulting in excision of the hpt gene. The recombination event could be detected because it places the CaMV 35S promoter of the hpt gene adjacent to a promoterless gusA gene; as a result the gusA gene is activated and its expression could be visualized. In 73 Cre/lox hybrid plants from various crosses of T0 transgenic plants, 19 expressed GUS, and in 132 Cre/lox hybrid plants from crosses of T2 transgenic plants, 77 showed GUS expression. Molecular data proved the excision event occurred in all the GUS⁺ plants. Recombination occurred with high efficiency at the early germinal stage, or randomly during somatic development stages. Received. 2 April 2001 / Accepted: 29 June 2001