Anatomy of herpes simplex virus DNA VII. alpha-RNA is homologous to noncontiguous sites in both the L and S components of viral DNA.

Previous reports from this laboratory (Honess and Roizman, 1974) have operationally defined alpha polypeptides as the viral proteins that are synthesized first in HEp-2 cells treated with cycloheximide from the time of infection with herpes simplex virus type 1 until the withdrawal of the drug 12 to 15 h after infection. It has also been shown that the viral RNA (designated alpha RNA) that accumulates in the cytoplasm during cycloheximide treatment and on polyribosomes immediately upon withdrawal of the drug is homologous to 10 to 12% of viral DNA, whereas the viral RNA accumulating in the cytoplasm of untreated cells at 8 to 14 h after infection is homologous to 43% of viral DNA (Kozak and Roizman, 1974). In the present study, alpha RNA and cytoplasmic RNA extracted from untreated cells 8 h after infection were each hybridized in liquid to in vitro labeled restriction endonuclease fragments generated by cleavage of herpes simplex virus type 1 DNA with Hsu I, with Bgl II, and with both enzymes simultaneously. The data show that only a subset of the fragments hybridized to alpha RNA, and these are scattered within both the L and S components of the DNA. There are at least five noncontiguous regions in the DNA homologous to alpha RNA; two of these are located partially within the reiterated sequences in the S component. All fragments tested hybridized more extensively with 8-h cytoplasmic RNA than with alpha RNA. Four adjacent fragments, corresponding to 30% of the DNA and mapping within the L component, hybridized exclusively with the cytoplasmic RNA extracted from cells 8 h after infection.     

Generation of an inverting herpes simplex virus 1 mutant lacking the L-S junction a sequences, an origin of DNA synthesis, and several genes including those specifying glycoprotein E and the alpha 47 gene.

The herpes simplex virus genome consists of two components, L and S, that invert relative to each other to yield four isomeric arrangements, prototype (P), inversion of the S component (Is), inversion of the L component (Il), and inversion of both components (Isl). Previous studies have shown that the 500-base-pair a sequences flanking the two components contain a cis-acting site for inversion. In an attempt to insert a third copy of the alpha 4 gene, the major regulatory gene mapping in the repeats flanking the S component, a fragment containing the alpha 4 gene and an origin of DNA synthesis, was recombined into the thymidine kinase gene mapping in the unique sequences of the L component. The resulting recombinants showed massive rearrangements and deletions mapping in the S component and in the junction between the L and S components. One recombinant (R7023) yielded two isomeric DNA arrangements, a major component consisting of Is and a minor component consisting of Isl. In these arrangements, the genome lacked the gene specifying glycoprotein E and all contiguous genes located between it and the alpha 0 gene in the inverted repeats of the L component. Among the deleted sequences were those encoding an origin of viral DNA synthesis, the alpha 47 gene, and the a sequences located at the junction between the L and S-components. The recombinant grew well in rabbit skin, 143TK-, and Vero cell lines. We conclude that the four unique genes deleted in R7023 are not essential for the growth of herpes simplex virus, at least in the cell lines tested, and that the b sequence of the inverted repeats of the L component also contains cis-acting sites for the inversion of herpes simplex virus DNA sequences.     

Herpes simplex virus amplicon: cleavage of concatemeric DNA is linked to packaging and involves amplification of the terminally reiterated a sequence.

Herpes simplex virus-infected cells contain large concatemeric DNA molecules arising from replication of the viral genome. The large concatemers are cleaved to generate unit-length molecules terminating at both ends with the a sequence. We have used constructed defective virus vectors (amplicons) derived from herpes simplex virus to study the mechanism of cleavage of viral DNA concatemers and the packaging of viral DNA into nucleocapsids. These studies revealed that (i) a 248-base-pair a sequence contained the signal(s) required for cleavage-packaging, (ii) the cleavage of viral DNA concatemers was coupled to packaging, (iii) the a sequence contained the information required for its own amplification, and (iv) cleavage-packaging occurred by a novel process involving the amplification of the a sequence.     

Functional domains within the a sequence involved in the cleavage-packaging of herpes simplex virus DNA.

Newly replicated herpes simplex virus (HSV) DNA consists of head-to-tail concatemers which are cleaved to generate unit-length genomes bounded by the terminally reiterated a sequence. Constructed defective HSV vectors (amplicons) containing a viral DNA replication origin and the a sequence are similarly replicated into large concatemers which are cleaved at a sequences punctuating the junctions between adjacent repeat units, concurrent with the packaging of viral DNA into nucleocapsids. In the present study we tested the ability of seed amplicons containing specific deletions in the a sequence to become cleaved and packaged and hence be propagated in virus stocks. These studies revealed that two separate signals, located within the Ub and Uc elements of the a sequence, were essential for amplicon propagation. No derivative defective genomes were recovered from seed constructs which lacked the Uc signal. In contrast, propagation of seed constructs lacking the Ub signal resulted in the selection of defective genomes with novel junctions, containing specific insertions of a sequences derived from the helper virus DNA. Comparison of published sequences of concatemeric junctions of several herpesviruses supported a uniform mechanism for the cleavage-packaging process, involving the measurement from two highly conserved blocks of sequences (pac-1 and pac-2) which were homologous to the required Uc and Ub sequences. These results form the basis for general models for the mechanism of cleavage-packaging of herpesvirus DNA.     

The herpes simplex virus 1 UL15 gene encodes two proteins and is required for cleavage of genomic viral DNA.

Previous studies have shown that a ts mutant [herpes simplex virus 1 (mP)ts66.4] in the UL15 gene fails to package viral DNA into capsids (A. P. W. Poon and B. Roizman, J. Virol. 67:4497-4503, 1993) and that although the intron separating the first and second exons of the UL15 gene contains UL16 and UL17 open reading frames, replacement of the first exon with a cDNA copy of the entire gene does not affect viral replication (J.D. Baines, and B. Roizman, J. Virol. 66:5621-5626, 1992). We report that (i) a polyclonal rabbit antiserum generated against a chimeric protein consisting of the bacterial maltose-binding protein fused in frame to the majority of sequences contained in the second exon of the UL15 gene reacted with two proteins with M(r) of 35,000 and 75,000, respectively, in cells infected with a virus containing the authentic gene yielding a spliced mRNA or with a virus in which the authentic UL15 gene was replaced with a cDNA copy. (ii) Insertion of 20 additional codons into the C terminus of UL15 exon II caused a reduction in the electrophoretic mobility of both the apparently 35,000- and 75,000-M(r) proteins, unambiguously demonstrating that both share the carboxyl terminus of the UL15 exon II. (iii) Accumulation of the 35,000-M(r) protein was reduced in cells infected and maintained in the presence of phosphonoacetate, an inhibitor of viral DNA synthesis. (iv) The UL15 proteins were localized in the perinuclear space at 6 h after infection and largely in the nucleus at 12 h after infection. (v) Viral DNA accumulating in cells infected with herpes simplex virus 1(mP)ts66.4 and maintained at the nonpermissive temperature was in an endless (concatemeric) form, and therefore UL15 is required for the cleavage of mature, unit-length molecules for packaging into capsids.     

Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.

We examined the fate of DNA microinjected into nuclei of cultured mammalian cells. The sequence composition and the physical form of the vector carrying the selectable gene affected the efficiency of DNA-mediated transformation. Introduction of sequences near the simian virus 40 origin of DNA replication or in the long terminal repeat of avian sarcoma provirus into a recombinant plasmid containing the herpes simplex virus thymidine kinase gene. (pBR322/HSV-tk) enhanced the frequency of transformation of LMtk- and RAT-2tk- cells to the TK+ phenotype 20- to 40-fold. In cells receiving injections of only a few plasmid DNA molecules, the transformation frequency was 40-fold higher after injection of linear molecules than after injection of supercoiled molecules. By controlling the number of gene copies injected into a recipient cell, we could obtain transformants containing a single copy or as many as 50 to 100 copies of the selectable gene. Multiple copies of the transforming gene were not scattered throughout the host genome but were integrated as a concatemer at one or a very few sites in the host chromosome. Independent transformants contained the donated genes in different chromosomes. The orientation of the gene copies within the concatemer was not random; rather, the copies were organized as tandem head-to-tail arrays. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, we were able to conclude that the head-to-tail concatemers were generated predominantly by homologous recombination. Surprisingly, these head-to-tail concatemers were found in transformants obtained by injecting either supercoiled or linear plasmid DNA. Even though we demonstrated that cultured mammalian cells contain the enzymes for ligating two DNA molecules very efficiently irrespective of the sequences or topology at their ends, we found that even linear plasmid DNA was recruited into the concatemer by homologous recombination.     

Herpes simplex virus 1 recombinants with noninverting genomes frozen in different isomeric arrangements are capable of independent replication.

Herpes simplex virus 1 genome consists of two covalently linked components, L and S, that invert relative to each other to yield four equimolar isomeric populations designated P (prototype), Is (inversion of S component), Il (inversion of L component), and Ils (inversion of L and S components) differing in the orientation of the two components. Previous studies have yielded recombinant genomes frozen in the P isomeric arrangement, reinforcing suggestions that the four isomers may not be functionally equivalent. We report the isolation of recombinants produced by insertional mutagenesis with alpha TK mini-Mu that are frozen in Is and Ils arrangements. Thus, all isomeric forms of herpes simplex virus DNA appear to be capable of independent replication and must be considered as functionally equivalent.     

Construction and properties of a recombinant herpes simplex virus 1 lacking both S-component origins of DNA synthesis.

The herpes simplex virus 1 (HSV-1) genome contains three origins of DNA synthesis (Ori) utilized by viral DNA synthesis proteins. One sequence (OriI) maps in the L component, whereas two sequences (OriS) map in the S component. We report the construction of a recombinant virus, R7711, from which both OriS sequences have been deleted, and show that the OriS sequences are not essential for the replication of HSV-1 in cultured cells. In addition to the deletions of OriS in R7711, the alpha 47 gene and the 5' untranscribed and transcribed noncoding regions of the U(S)11 gene were deleted, one of the alpha 4 promoter-regulatory regions was replaced with the simian virus 40 promoter, and the alpha 22 promoter was substituted with the alpha 27 promoter. The total amount of viral DNA synthesized in Vero cells infected with the OriS-negative (OriS-) virus was approximately that seen in cells infected with the OriS-positive virus. However, cells infected with the OriS- virus accumulated viral DNA more slowly than those infected with the wild-type virus during the first few hours after the onset of DNA synthesis. In single-step growth experiments, the yield of OriS- progeny virus was reduced at most fourfold. Although a single OriS (R. Longnecker and B. Roizman, J. Virol. 58:583-591, 1986) and the single OriL (M. Polvino-Bodnar, P. K. Orberg, and P. A. Schaffer, J. Virol. 61:3528-3535, 1987) have been shown to be dispensable, this is the first indication that both copies of OriS are dispensable and that one copy of an Ori sequence may suffice for the replication of HSV-1.     

Characterization of ICP6::lacZ insertion mutants of the UL15 gene of herpes simplex virus type 1 reveals the translation of two proteins.

The herpes simplex virus type 1 (HSV-1) UL15 gene is a spliced gene composed of two exons and is predicted to encode an 81-kDa protein of 735 amino acids (aa). Two UL15 gene products with molecular masses of 75 and 35 kDa have been observed (J. Baines, A. Poon, J. Rovnak, and B. Roizman, J. Virol. 68:8118-8124, 1994); however, it is not clear whether the smaller form represents a proteolytic cleavage product of the larger form or whether it is separately translated. In addition, an HSV-1 temperature-sensitive mutant in the UL15 gene (ts66.4) is defective in both cleavage of viral DNA concatemers into unit-length monomers and packaging of viral DNA into capsids (A. Poon and B. Roizman, J. Virol. 67:4497-4503, 1993; J. Baines et al., J. Virol. 68:8118-8124, 1994). In this study, we detected two UL15 gene products of 81 and 30 kDa in HSV-1-infected cells, using a polyclonal antibody raised against a maltose binding protein fusion construct containing UL15 exon 2. In addition, we report the isolation of two HSV-1 insertion mutants, hr81-1 and hr81-2, which contain an ICP6::lacZ insertion in UL15 exon 1 and exon 2 and thus would be predicted to encode C-terminally truncated peptides of 153 and 509 aa long, respectively. hr81-1 and hr81-2 are defective in DNA cleavage and packaging and accumulate only B capsids. However, both mutants are able to undergo wild-type levels of DNA replication and genomic inversion, suggesting that genomic inversion is a result of DNA replication rather than of DNA cleavage and packaging. We also provide evidence that the 81- and 30-kDa proteins are the products of separate in-frame translation events from the UL15 gene and that the 81-kDa full-length UL15 protein is required for DNA cleavage and packaging.     

DNA polymerase in nuclei isolated from herpes simplex virus type-2-infected cells. Characterization of the reaction product and inhibition by substrate analogs

Nuclei isolated from herpes simplex virus (HSV) type 2-infected KB cells were examined for their capacity to serve as an in situ source of herpes DNA polymerase. In contrast to purified enzymes with added template, approx. 80% of the DNA synthesized in isolated nuclei was viral. The average size of DNA fragments labeled in vitro was 3.2 X 10(6) Da. Based on an increase in DNA density when nuclei were incubated in the presence of BrdUTP rather than dTTP, 16% of the nucleotides were added during the in vitro reaction. Sucrose gradient analysis of DNA polymerase activity in extracts of isolated nuclei demonstrated the nearly exclusive presence of herpes DNA polymerase. Km concentrations for the four dNTPs were from 0.14 to 0.55 microM. DNA synthesis was inhibited competitively by the 5'-triphosphates of ara-A and ara-C (Ki = 0.03 and 0.22 microM, respectively) but not by the 5'-triphosphate of dideoxythymidine. aATP also served as a substrate (Km = 0.014 microM) for the reaction. We conclude that nuclei from HSV-infected cells have significant advantages for the detailed study of inhibitors of herpesvirus replication.     

A noninverting genome of a viable herpes simplex virus 1: presence of head-to-tail linkages in packaged genomes and requirements for circularization after infection.

The wild-type herpes simplex virus 1 genome consists of two components, L and S, which invert relative to each other, giving rise to four isomers. Previously we reported the construction of a herpes simplex virus 1 genome, HSV-1(F)I358, from which 15 kilobase pairs of DNA spanning the junction between L and S components were deleted and which no longer inverted (Poffenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2690-2694, 1983). Further studies on the structure of HSV-1(F)I358 revealed the presence of two submolar populations among packaged DNA. The first, comprising no more than 10% of total packaged DNA, consisted of defective genomes with a subunit size of 36 kilobase pairs. The results suggest that this population arose by recombination through a directly repeated sequence inserted in place of the deleted L-S junction. The second minor population consisted of HSV-1(F)I358 DNA linked head-to-tail. Analyses of the structure of HSV-1(F)I358 DNA after infection indicated that the fraction of total DNA linked head-to-tail increased to approximately 40 to 50% within 30 min after exposure of cells to virus. The formation of head-to-tail linkages did not require de novo protein synthesis. Our interpretation of the results is that the termini of full-length DNA molecules are held together during packaging, that a small fraction of the termini is covalently linked during or after packaging, and that the remainder is covalently joined after the release of viral DNA from the infecting virus by either host or viral factors introduced into the cell during infection.     

Redistribution of microtubules and Golgi apparatus in herpes simplex virus-infected cells and their role in viral exocytosis.

Earlier studies have shown that the Golgi apparatus was fragmented and dispersed in herpes simplex virus 1-infected Vero and HEp-2 cells but not in human 143TK- cells, that the fragmentation and dispersal required viral functions expressed concurrently with or after the onset of DNA synthesis (G. Campadelli-Fiume, R. Brandimarti, C. Di Lazzaro, P. L. Ward, B. Roizman, and M. R. Torrisi, Proc. Natl. Acad. Sci. USA 90:2798-2802, 1993), and that in 143TK- cells, but not Vero or HEp-2 cells, infected with viral mutants lacking the UL20 gene virions were glycosylated and transported to extracellular space (J. D. Baines, P. L. Ward, G. Campadelli-Fiume, and B. Roizman, J. Virol. 65:6414-6424, 1991; E. Avitabile, P. L. Ward, C. Di Lazzaro, M. R. Torrisi, B. Roizman, and G. Campadelli-Fiume, J. Virol. 68:7397-7405, 1994). Experiments designed to elucidate the role of the microtubules and of intact or fragmented Golgi apparatus in the exocytosis of virions showed the following. (i) In all cell lines tested (Vero, 143TK-, BHK, and Hep-2) microtubules underwent fragmentation particularly evident at the cell periphery and then reorganized into bundles which circumvent the nucleus. This event was not affected by inhibitors of viral DNA synthesis. We conclude that redistribution of microtubules may be required but is not sufficient for the fragmentation and dispersal of the Golgi apparatus. (ii) In all infected cell lines tested, nocodazole caused fragmentation and dispersal of the Golgi and a far more extensive depolymerization of the microtubules than was seen in untreated, infected Vero or HEp-2 cells. Taxol precluded the depolymerization of the microtubules and fragmentation of the Golgi in both infected cell lines. Neither nocodazole nor taxol affected the exocytosis of infectious virus from Vero, HEp-2, or 143TK- cells infected with wild-type virus. We conclude that the effects of nocodazole or of taxol are dominant over the effects of viral infection in the cell lines tested and that viral exocytosis is independent of the organization of microtubules or of the integrity of the Golgi apparatus. Lastly, the data suggest that herpes simplex viruses have evolved an exocytic pathway for which the UL20 protein is a component required in some cells but not others and in which this protein does not merely compensate for the fragmentation and dispersal of the Golgi apparatus.     

Molecular genetics of herpes simplex virus. VIII. further characterization of a temperature-sensitive mutant defective in release of viral DNA and in other stages of the viral reproductive cycle.

Previous studies have shown that cells infected with the herpes simplex virus 1(HFEM) mutant tsB7 and maintained at the nonpermissive temperature fail to accumulate viral polypeptides. Analyses of intertypic recombinants generated by marker rescue of tsB7 with herpes simplex virus 2 DNA fragments localized the mutation between 0.46 and 0.52 map units on the viral genome (Knipe et al., J. Virol. 38:539-547, 1981). In this paper we report that the mutation in tsB7 affects several aspects of the reproductive cycle of the virus at the nonpermissive temperature. Thus, (i) viral capsids accumulate at the nuclear pores and do not release viral DNA for at least 6 h postinfection at 39 degrees C. The DNA was released within 30 min after a shift to the permissive temperature. (ii) Experiments involving shifts from the permissive to the nonpermissive temperature indicated that viral protein synthesis was not sustained in cells maintained at the permissive temperature for less than 4 h. (iii) Viral DNA synthesis was delayed at the permissive temperature for as long as 8 h. Once initiated, it continued at 39 degrees C. (iv) Marker rescue of tsB7 by transfection with herpes simplex virus 1(F) DNA fragments localized the mutation to between 0.501 and 0.503 map units on the viral genome. These results are consistent with the tsB7 lesion being in a gene coding for a virion component which affects release of viral DNA from capsids and onset of viral DNA synthesis.     

Adenovirus type 2 DNA replication. I. Evidence for discontinuous DNA synthesis.

Isolated nuclei from adenovirus type 2-infected HeLa cells catalyze the incorporation of all four deoxyribonucleoside triphosphates into viral DNA. The observed DNA synthesis occurs via a transient formation of DNA fragments with a sedimentation coefficient of 10S. The fragments are precursors to unit-length viral DNA, they are self-complementary to an extent of at least 70%, and they are distributed along most of the viral chromosome. In addition, accumulation of 10S DNA fragments is observed either in intact, virus-infected HeLa cells under conditions where viral DNA synthesis is inhibited by hydroxyurea or in isolated nuclei from virus-infected HeLa cells at low concentrations of deoxyribonucleotides. Under these suboptimal conditions for DNA synthesis in isolated nuclei, ribonucleoside triphosphates determine the size distribution of DNA intermediates. The evidence presented suggests that a ribonucleoside-dependent initiation step as well at two DNA polymerase catalyzed reactions are involved in the discontinuous replication of adenovirus type 2 DNA.     

Detection of herpes simplex virus-specific DNA sequences in latently infected mice and in humans.

Herpes simplex virus-specific DNA sequences have been detected by Southern hybridization analysis in both central and peripheral nervous system tissues of latently infected mice. We have detected virus-specific sequences corresponding to the junction fragment but not the genomic termini, an observation first made by Rock and Fraser (Nature [London] 302:523-525, 1983). This "endless" herpes simplex virus DNA is both qualitatively and quantitatively stable in mouse neural tissue analyzed over a 4-month period. In addition, examination of DNA extracted from human trigeminal ganglia has shown herpes simplex virus DNA to be present in an "endless" form similar to that found in the mouse model system. Further restriction enzyme analysis of latently infected mouse brainstem and human trigeminal DNA has shown that this "endless" herpes simplex virus DNA is present in all four isomeric configurations.     

Conservation and progressive methylation of Epstein-Barr viral DNA sequences in transformed cells.

The structure of intracellular viral DNA from a number of cell lines arising by clonal transformation of human lymphocytes in vitro with Epstein-Barr virus was analyzed. Intracellular viral DNAs were partially purified and digested with several restriction endonucleases, and the products of digestion were separated by electrophoresis in agarose gels. The viral fragments were detected by transferring the DNA from the gel to nitrocellulose sheets, hybridizing radiolabeled recombinant vectors carrying fragments of viral DNA to those transfers, and visualizing the hybrids by autoradiography. These analyses indicated that: (i) regions of repetitious viral DNA do undergo expansion and contraction although one size predominates; (ii) novel sequence arrangements appear in the intracellular viral DNA of different clones but are not found in clones analyzed serially and propagated extensively; (iii) the viral DNA is increasingly methylated upon cell propagation. We have not identified a transformed cell phenotype or a viral phenotype that segregates with the observed progressive methylation. We have not detected in Epstein-Barr viral plasmids analogs of the gross rearrangements of viral DNAs observed after lytic infections with high multiplicities of papova-, adeno-, or herpes simplex viruses.     

A temperature-sensitive mutation in a herpes simplex virus type 1 gene required for viral DNA synthesis maps to coordinates 0.609 through 0.614 in UL

ts701 is a temperature-sensitive mutant of herpes simplex virus type 1 strain KOS induced by hydroxylamine mutagenesis (C.T. Chu, D. S. Parris, R. A. F. Dixon, F. E. Farber, and P. A. Schaffer, Virology 98:168-181, 1979). In the present study, the mutation rendering ts701 temperature sensitive was mapped to coordinates 0.609 through 0.614 in the UL region of the genome. At the nonpermissive temperature, ts701 (i) failed to induce the synthesis of viral DNA, (ii) exhibited a dramatically reduced ability to drive replication of a plasmid containing the herpes simplex virus origin of viral DNA synthesis, oriS, (iii) generated no viral polypeptides of the late (gamma 2) kinetic class, and (iv) produced virions with electron-translucent cores. Northern (RNA) blot hybridization demonstrated that two mRNAs--one of the beta kinetic class and one of the gamma kinetic class--hybridized to a 1.3-kilobase viral DNA fragment that rescued the mutation in ts701. Based on the phenotype and mapping of ts701, it is likely that its mutation lies in the gene specifying the 65,000-Mr DNA-binding protein (65KDBP) recently described by Marsden et al. (H.S. Marsden, M.E.M. Campbell, L. Haarr, M. C. Frame, D. S. Parris, M. Murphy, R. G. Hope, M. T. Muller, and C. M. Preston, J. Virol. 61:2428-2437, 1987).     

Location of the structural genes for glycoproteins gD and gE and for other polypeptides in the S component of herpes simplex virus type 1 DNA.

To map the structural genes for the gD and gE polypeptides and for other viral products encoded in the S component of herpes simplex virus type 1 DNA, we selected mRNAs capable of hybridizing to cloned viral DNA fragments and translated the mRNAs in vitro to determine which polypeptides were encoded therein. The gD and gE polypeptides were identified by immunoprecipitation with appropriate monoclonal and monospecific antibodies, whereas the other polypeptides were characterized only by their electrophoretic mobilities in polyacrylamide gels. We found that gD mRNA hybridized to a single SacI subfragment of BamHI fragment J, whereas gE mRNA hybridized to an adjacent SacI subfragment of BamHI fragment J and also to BamHI fragment X. These and other results permit the conclusion that the structural gene for gD is located between map coordinates 0.911 and 0.924, and the gene for gE is between map coordinates 0.924 and 0.951. We also found that mRNAs for polypeptides of 55,000, 42,000, 33,000, and 22,000 molecular weight hybridized to DNA fragments spanning the regions from map coordinates 0.911 to 0.924, 0.897 to 0.911, 0.939 to 0.965, and 0.939 to 0.965, respectively. Finally, in accord with the results of others, we found that mRNA for a 68,000-molecular-weight polypeptide hybridized to the two noncontiguous BamHI fragments N and Z, which share a reiterated DNA sequence.