The kinetics of adenovirus recombination in homotypic and heterotypic genetic crosses

The kinetics of recombination between temperature-sensitive mutants has been studied in both homotypic (Ad5 x Ad5) and heterotypic (Ad5 x Ad2+NDI) crosses. There is a significant increase in the recombination frequency during the rise period of viral replication in a single-step replication cycle. This observation suggests that adenovirus DNA molecules can undergo progressive rounds of recombination before assembly into virions. To examine this possibility further, advantage was taken of the serotype-specific restriction endonuclease sites and polypeptide sizes of Ad5 and Ad2+ND1 to enumerate and to localize the sites of crossing over in ts+ recombinants isolated early and late in a heterotypic infection. The late recombinants exhibited, on average, more crossovers per genome than did the early recombinants. This finding is predicted if multiple rounds of recombination take place in some genomes. Using blot hybridization with specific probes, the production of recombinant molecules in the intracellular DNA replicating pool has been followed. Recombinants were found before the rise in infectious virus and increased in frequency relative to the parental molecules throughout the exponential period. These data confirm and extend the genetic observations, which were made on a selected set of infectious virus.     

A genetic recombination map of foot-and-mouth disease virus.

Sixty ts mutants were isolated from the Pacheco strain of type O foot-and-mouth disease virus after treatment with either 5-fluorouracil or hydroxylamine. The conditions affecting recombination and assay of the ts+ recombinants were standardized. Using two ts mutants resistant to guanidine, three-factor crosses, supported by two-factor crosses, located 34 of the mutations in a linear arrangement. The recombination frequencies between certain pairs of mutations were additive. The guanidine character of the two resistant mutants mapped as a single site mutation and was located near the middle of the map.     

Mapping of base sequence heterologies between genomes from different adenovirus serotypes

An electron microscopic heteroduplex technique was used to physically map sequence heterologies between genomes from different human adenovirus serotypes. When DNA from serotypes was mixed, denatured, and renatured, characteristic heteroduplex molecules were formed. These molecules, composed of one strand from one serotype and one strand from the other serotype, contained one or more detectable regions of strand separation (or heterology), depending upon denaturing conditions and the combination of type-specific DNA preparations. By increasing denaturing conditions, regions of partial homology could be demonstrated in all interserotypic heteroduplexes. The heterology patterns in heteroduplexes constructed with DNA from nononcogenic serotypes (Ad 1,2, and 5) were relatively simple and nearly similar, whereas both simple and complex patterns were seen in heteroduplexes prepared with DNA from weakly oncogenic serotypes (Ad3,7, and 21). Only complex heterology patterns were found in heteroduplexes prepared with DNA from highly oncogenic serotypes (Ad 12, 18, and 31). As expected, the most extensive heterologies were observed in heteroduplexes prepared with DNA from serotypes belonging to the different serologic groups. Within groups, heteroduplex molecules contained two specific regions in which heterologies were most marked. These regions appeared to map at similar locations among heteroduplexes from all groups. It was found that the integrated SV40 virus DNA in an Ad 7-SV40 hybrid genome was situated in one of these regions.     

Multiple recombination sites at the 5'-end of murine coronavirus RNA

Mouse hepatitis virus (MHV), a murine coronavirus, contains a nonsegmented RNA genome. We have previously shown that MHV could undergo RNA-RNA recombination in crosses between temperature-sensitive mutants and wild-type viruses at a very high frequency (S. Makino, J.G. Keck, S.A. Stohlman, and M.M.C. Lai (1986) J. Virol. 57, 729-737). To better define the mechanism of RNA recombination, we have performed additional crosses involving different sets of MHV strains. Three or possibly four classes of recombinants were isolated. Recombinants in the first class, which are similar to the ones previously reported, contain a single crossover in either gene A or B, which are the 5'-most genes. The second class of recombinants contain double crossovers in gene A. The third class of recombinants have crossovers within the leader sequence located at the 5'-end of the genome. The crossover sites of the third class have been located between 35 and 60 nucleotides from the 5'-end of the leader RNA. One of these recombinants has double crossovers within the short region comprising the leader sequences. Finally, we describe one recombinant which may contain a triple crossover. The presence of so many recombination sites within the 5'-end of the genome of murine coronaviruses confirms that RNA recombination is a frequent event during MHV replication and is consistent with our proposed model of "copy-choice" recombination in which RNA replication occurs in a discontinuous and nonprocessive manner.     

Recombination in adenovirus: DNA sequence analysis of crossover sites in intertypic recombinants

The nucleotide sequence of the adenovirus type 5 genome has been determined for a 620-bp region that spans the C terminus of the pVI gene and the N terminus of the hexon gene, and compared to the adenovirus type 2 DNA sequence: 25 base changes have been identified, most of which do not lead to alterations in the amino acid sequence and regulatory signals in the region. Crossover sites in three intertypic recombinants have been previously located in this region of the genome by fine restriction mapping. A sequence determination for the three recombinants, and the four ts mutants used in generating the ts+ recombinants, was carried out. The crossovers were in each case located in a small region of complete sequence homology (from 45 to 156 nucleotides long) flanked on either side by sequences derived from each parent. These structures are compatible with a reciprocal crossing over model of generalised recombination, where a recombinant joint has resolved in a region of high DNA homology. For the recombinants considered here, this region abutts onto a neighbouring region of much lower sequence homology, and it is possible that the position of the crossover is determined at least in part by the termination of branch migration at a heterologous boundary.     

Multiple sites of recombination within the RNA genome of foot-and-mouth disease virus

Recombinant foot-and-mouth disease viruses were isolated from cells infected with a mixture of temperature-sensitive (ts) mutants belonging to different subtype strains. In order to select for recombination events in many different regions of the genome, crosses were performed between various pairs of mutants, with ts mutations in different regions of the genome. ts+ progeny were analysed by electrofocusing virus-induced proteins and RNase T1 fingerprinting of their RNA. All but 5 out of 43 independent isolates, from nine crosses, proved to have recombinant RNA genomes. Maps of these genomes, based on a knowledge of the locations of the unique oligonucleotides, were constructed. Most could be interpreted as being the products of single genetic cross-overs, although three recombinants were formed by two cross-overs each. Cross-overs in at least twelve distinct regions of the genome were identified. This evidence of a large number of recombination sites suggests that RNA recombination in picornaviruses is a general, as opposed to a site-specific, phenomenon.     

Temperature-sensitive mutants of herpes simplex virus type 2: a provisional linkage map based on recombination analysis.

Thirteen ts mutants of type 2 herpes simplex virus were backcrossed to a syncytial but not temperature-sensitive mutant of wild-type virus. This was an attempt to introduce a third marker, syncytial plaque morphology or syn, into at least some of the ts mutants. Three ts mutants carrying the syn marker were obtained but only one, ts 9, was satisfactory for genetic experiments. Three-factor crosses were carried out between ts 9 syn and the mutants which determined the order of eleven ts mutations relative to both the ts 9 mutation and the syn mutation. A provisional linkage map based both on the order derived from the three-factor crosses and on map distances from recombination frequencies has been prepared: it contains nine ts mutations and the syn mutation.     

Recombination between temperature-sensitive mutants of herpes simplex virus type 1

Fifteen temperature-sensitive (ts) mutants of herpes simplex virus type 1 representing 10 complementation groups were examined for their ability to recombine. Efficient recombination was demonstrated between most mutant pairs in standard two-factor crosses. Mutants which failed to complement each other also failed to recombine or recombined with low frequency. Progeny analysis of putative ts⁺ recombinants demonstrated that complementing clumps and/or multiploid particles did not contribute significantly to recombinant yields. Using recombination data from crosses between 11 mutants representing 7 complementation groups, a provisional linkage map has been constructed. The map spans approximately 38 recombination units and is linear.     

Physical and genetic analysis of the herpes simplex virus DNA polymerase locus

The physical location of the mutations tsC7, tsC4, and tsD9 (strain KOS), all of which affect HSV DNA polymerase, have been located on the physical map of the HSV-1 genome. Intertypic recombinants between HSV-1 mutants tsC4, tsD9, and tsC7 and HSV-2 (strain HG52) were generated by marker rescue. Restriction endonuclease analysis using five restriction enzymes led to the identification of the parental origin of the DNA sequences in the genomes of 15 recombinants of HSV-1 tsD9 with HSV-2, 13 recombinants of HSV-1 tsC7 with HSV-2, and 12 recombinants of HSV-1 tsC4 with HSV-2. In addition to the ts marker used for selection, a nonselected marker (phosphonoacetic acid resistance, paa^r) was present in all the crosses. All the mutations cluster in a 4.9-kilobase pair (kbp) region between map units 38.6 to 41.8. The mutations for paa^r and tsD9 map within a 2.9-kbp region (40 to 41.8 map units). The tsC7 mutation is contained in a 1.5-kbp region to the left of the other (map units 38.6 to 39.6). The mapping limits for the mutation tsC4 are between map units 39.3 and 41.8. The mutations, at this point, have not been assigned to specific genes but the physical mapping results presented in this study clearly show clustering of these mutations in a discrete region of the genome.     

Heterologous recombination between Autographa californica nuclear polyhedrosis virus DNA and foreign DNA in non-polyhedrin segments of the viral genome

We used the expression vector system of Autographa californica nuclear polyhedrosis virus (AcNPV) and Spodoptera frugiperda insect cells to study mechanisms of recombination in insect cells. We concentrated on the isolation and analysis of heterologous recombinants. The E1 region of human adenovirus type 2 (Ad2) was inserted into regions of the AcNPV genome which lacked apparent homologies to the polyhedrin region. Out of a total of 122 recombinant AcNPV plaques, which hybridized to Ad2 DNA in plaque annealing experiments, 13 recombinants proved heterologous, and 5 of these recombinants could be grown to titers that facilitated virus replication and further investigations of the recombinant DNA. Restriction and Southern blot analyses for all of the recombinants and nucleotide sequence determinations for one of them permitted the mapping of the sites of foreign DNA integration into the AcNPV genome for the heterologous recombinants. These sites were located in the EcoRI-C (map units 42.5-52.4), the EcoRI-L (map units 69.5-72.5), the EcoRI-O (map units 32.6-34.5), and the EcoRI-Q (map units 88.2-89.7) segments of the plaque isolate E AcNPV genome. Two of the heterologous recombinants carried the insert in the EcoRI-L fragment. The nucleotide sequence determinations across the sites of junction between the AcNPV DNA and the foreign (Ad2) DNA in one of the heterologous recombinants, AcNPV-Ad2E1-D, revealed no sequence similarities at or close to the sites of junctions. A short sequence of six nucleotides was deleted from the original EcoRI-O sequence of AcNPV at the site of insertion. The inserted Ad2E1 DNA fragment comprised nucleotides 183-2763; thus nucleotides at the termini had been deleted. In the usual polyhedrin gene-located recombinants, the foreign Ad2 DNA segment was fused to the polyhedrin promoter and recombined presumably via polyhedrin sequence segments in the vector into the polyhedrin gene of AcNPV. In one of the control recombinants, AcNPV-Ad2E1-192, the Ad2E1 DNA segment between nucleotides 1 and 3117 (out of 3322 original nucleotides) was inserted in an inverted orientation between nucleotides -115 and +735 of the polyhedrin gene of AcNPV. This particular polyhedrin sequence was deleted in the process. It was uncertain how this recombinant had been generated. The infectivities of the polyhedrin-located recombinant AcNPV-Ad2E1-192 and of the five heterologous recombinants were compared by single-cycle growth curves to the infectivity of non-recombinant AcNPV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Assignment of simian rotavirus SA11 temperature-sensitive mutant groups A, C, F, and G to genome segments

Crosses were performed between prototype temperature-sensitive (ts) mutants of simian rotavirus SA11 representing reassortment groups A, C, F, and G and ts mutants of rhesus rotavirus RRV that belonged to different reassortment groups. Wild-type (ts+) reassortant progeny were identified by plaque formation at nonpermissive temperature (39 degrees), picked, and grown to high titer. The ts+ phenotype of the resulting progeny clones was verified by titration at 39 degrees and 31 degrees. The electropherotypes of the ts+ clones were determined by electrophoresis, and parental origin of each genome segment was assigned by comparison of segment mobility to parental markers. Analysis of the parental origin of genome segments in the ts+ reassortants derived from SA11 ts X RRV ts crosses revealed the following map locations of the SA11 prototype ts mutants: tsA(778), segment 4; tsC(606), segment 1; tsF(2124), segment 2; and tsG(2130), segment 6. The assignment of tsA was made on the basis of genome segment segregation in two independent crosses with each of two independent RRV ts mutants. The assignment of tsC was made on the basis of segregation in only a single cross with an RRV ts mutant; however, a larger number of progeny clones were examined from this cross. The lesion of tsF was mapped with data from three independent crosses using two different RRV ts mutants. The assignment of tsG was made on the basis of segregation in three independent crosses, two with RRV ts mutants and one with Wa. The assignments of tsA, tsC, and tsF were confirmed in crosses between RRV ts mutants representing those reassortment groups, and SA11 ts mutants in other reassortment groups.     

Recombination of parental lambda phages labeled by means of host-controlled modification

Two-factor genetic crosses with different configurations of the selected markers in λ · P and λ · K infecting phages were performed in Escherichia coli K12 bacteria. The frequency of λsus⁺ recombinants among the progeny phages that retained a parental λ · P HCM label was determined for crosses of λsus029 with λsusP3, of λsusP3 with λsusQ73 and of λsusQ73 with λsusR5. It was found that λ · P recombinants are formed not only in crosses of two λ · P phage types but can also be generated by single recombination of λ · P and λ · K genomes between susP3 and susQ73, or between susQ73 and susR5; only multiple crossovers between λ · P and λ · K genomes can give rise to λ · P recombinants for sus029 and susP3. The data indicate that the site of P1-mediated HCM that is nearest the right end of the λ chromosome is located between susP3 and susQ73, approximately 89% of the chromosome length from the left end of the physical map.     

乙型肝炎病毒表面抗原S基因在人5型重组腺病毒中的表达及其特性

The hepatitis B virus (HBV) S gene, PreS2 + S genes and the late phase expression cassette (MTI) + HBV S genes were separately cloned into Ad5 vector downstream of E3 promoter (pAd5 deltaE3 provided by Wyeth Co.). The above constructed plasmids and Ad5 DNA EcoR I A fragment were cotransfected into 293 cells. The progeny adenoviruses named rAd5S, rAd5MS, rAd5S2S were harvested for analysis. The recombinants were isolated and analyzed by PCR, using two primers specific to the HBV S genes. The expressed products were detected by ELISA and RIA. The recombinant containing MIT + HBV S genes (rAd5MS) was identified to be ELISA positive, whereas the other two recombinants (rAd5S, rAd5S2S) were negative to ELISA, but positive to RIA. The results indicated that adenovirus E3 early promoter could express the inserted foreign genes, and MIT worked well in the E3 region of Ad5 and could increase the expression capacity of the recombinants. The conditions for foreign gene expression and genetic stability of the recombinant viruses were studied in detail. There was no wild Ad5 discovered during the cotransfection experiments. The present study provides some experiences for studying adenovirus recombination.     

Studies on Recombination within the Mouse H-2 Complex I.: Three Recombinants Which Position the Ss Locus within the Complex

A series of genetic crosses conducted to map the Ss serum variant with respect to the H—2 complex is described in detail. Three intra- H—2 recombinants were detected. These crossovers, designated H—2 ^al, H—2 ^ol and H—2 ^tl, position the Ss gene in the middle of the H—2 complex between the regions H—2K and H—2D. The production of eight new congenic resistant inbred strains is also described.     

Genetic analysis of adenovirus type 2. IX. The physical locations of structural genes

Several virus-specified proteins of adenovirus type 2 (Ad2) and type 5 (Ad5) can be distinguished by their differential electrophoretic mobility in SDS-polyacrylamide gels. By comparing the physical maps of the genomes of interserotypic recombinants between ts mutants of these viruses with the pattern of proteins expressed, the physical locations of some genes can be determined on the adenovirus DNA molecule. The proteins of a total of 103 recombinants were analysed; 44 of these are of known DNA structure. Structural genes for the proteins analysed span the following loci on the conventional adenovirus map: V (30 to 43); III (43 to 44); II and 66K (55 to 57); 100K, 36K, 32K (69 to 71); IV (80 to 90).     

Strong correlation between meiotic crossovers and haplotype structure in a 2.5-Mb region on the long arm of chromosome 21

Although the haplotype structure of the human genome has been studied in great detail, very little is known about the mechanisms underlying its formation. To investigate the role of meiotic recombination on haplotype block formation, single nucleotide polymorphisms were selected at a high density from a 2.5-Mb region of human chromosome 21. Direct analysis of meiotic recombination by high-throughput multiplex genotyping of 662 single sperm identifies 41 recombinants. The crossovers were nonrandomly distributed within 16 small areas. All, except one, of these crossovers fall in areas where the haplotype structure exhibits breakdown, displaying a strong statistically positive association between crossovers and haplotype block breaks. The data also indicate a particular clustered distribution of recombination hotspots within the region. This finding supports the hypothesis that meiotic recombination makes a primary contribution to haplotype block formation in the human genome.     

Isolation and biochemical characterization of intertypic recombinants of foot-and-mouth disease virus

Recombinants were isolated between two European serotypes (O and A) and between two of the most distantly related serotypes (O from Europe and SAT2 from Africa) using appropriate ts mutants in an infectious centre assay. The recombinants were characterised by electrofocusing of their induced proteins and by RNase-T1 fingerprinting of their RNA. The approximate location of the cross-over event in each recombinant was determined by sequencing the unique distinguishable O or A oligonucleotides and locating them within the known genome sequence. Nine different types of recombinant were identified from the two types of cross (O X A and O X SAT) and all had a single cross-over in the middle or 3' half of the genome, i.e. in the nonstructural coding region. Recombination between the most distantly related viruses (O X SAT2) appeared to occur at a lower frequency than recombination between serotypes of the same group (O X A). A higher incidence of recombinant proteins with unique pI was also observed in the O X SAT2 crosses.     

Analysis of chickens for recombination within the MHC (B-complex)

In an attempt to further map the chicken MHC (the B complex), a systematic search for genetic recombinants within the B complex was performed by serotyping the progeny from F2 crosses of chickens by means of specific anti-class I, anti-class II, and anti-class IV alloantisera. Two recombinant B-haplotypes (B21r and B15r) were found by analysing 2,656 F2 chickens representing 5,312 informative typings. In either case, the B-G (class IV) allele was recombined with both the B-F and B-L alleles of the opposite haplotype. MLC typings, tests for direct compatibility by GVH reactions, and absorption analyses confirmed the original serological typing of the two recombinant B haplotypes. No recombination between B-F (class I) and B-L (class II) loci was found. This very low frequency of recombination within the B complex as compared with recombination frequencies found in mammalian MHC's is discussed.     

Multiple adjacent or overlapping loci affecting the level of gC and cell fusion mapped by intratypic recombinants of HSV-1

We have prepared and analyzed 40 HSV-1 intratypic recombinants with regard to plaque morphology and glycoprotein C(gC) phenotypes. Vero cells have been cotransfected with the intact genome of HSV-1(F) and cloned or uncloned DNA fragments from HSV-1(MP) and recombinants inducing the fusion of Vero cells [syncytial (Syn) recombinants] have been selected and purified. Marker transfer of the Syn phenotype has been observed with the cloned BamHI L and B fragments (0.706-0.745 and 0.745-0.810 map units, respectively) as well as with the uncloned HpaI TXO fragment (0.710-0.761) from MP DNA. No marker transfer has been observed with F DNA alone or with the cloned BamHI N fragment (0.863-0.898 map units). When viruses expressing the Syn phenotype in Vero cells were tested in HEp-2 cells, three kinds of recombinants were observed. Members of the first class expressed a wild type, cytoaggregating (Syn+), plaque morphology in these cells. Members of the second class induced the complete fusion (Syn phenotype) of the cells. Members of the third class induced an intermediate plaque morphology, characterized by the formation of groups of polykaryocytes (fused cells) but without formation of a complete syncytium. All recombinants expressing the Syn+ phenotype in HEp-2 cells were also gC+, whereas recombinants expressing the Syn phenotype in these cells were gC- with one exception, in which low levels of gC could be detected (but clearly less than with HSV-1(F]. Concerning polykaryocytic class of recombinants, some of them were gC+ while others expressed only low amounts of gC; no gC- virus was observed within this class of recombinants. The three classes of recombinants were observed with each of the cloned BamHI L and B fragments and also with the HpaI TXO fragment, suggesting the existence of multiple adjacent or overlapping loci affecting plaque morphology and the control of the accumulation or the synthesis of gC at both sides of 0.745 map units.     

Polyoma virus-transformed cells produce transforming growth factor(s) and grow in serum-free medium

Vaccinia virus temperature-sensitive (ts) mutants were isolated after nitrosoguanidine mutagenesis. A number of these mutants exhibited host range temperature sensitivity in that the efficiency of plaque formation at the nonpermissive temperature was poorer on chick cells than on hamster or human cells. Forty-two mutants were assigned to 23 different complementation groups on the basis of complementation and the efficiency of apparent recombination at the nonpermissive temperature. Recombination frequencies were also determined from mixed infections carried out at the permissive temperature and it was confirmed that mutants within the same complementation group recombined less efficiently with each other than mutants belonging to different groups. Mutants from two of the largest groups could be tentatively ordered on linear intragenic maps that spanned 0.8 and 2 recombination units. Moreover, from intergenic crosses between mutants in 14 different complementation groups, a linkage map spanning 66.3 recombination units, was derived. This study illustrates the feasibility of two-factor recombination mapping of poxvirus mutations and provides genetic data that should be of relevance in further analysis of the is mutations.