Showing papers on "Restriction map published in 1986"

Yeast/E. coli shuttle vectors with multiple unique restriction sites

Abstract: Two yeast/E. coli shuttle vectors have been constructed. The two vectors, YEp351 and YEp352, have the following properties: (1) they can replicate autonomously in Saccharomyces cerevisiae and in E. coli; (2) they contain the beta-lactamase gene and confer ampicillin resistance to E. coli; (3) they contain the entire sequence of pUC18; (4) all ten restriction sites of the multiple cloning region of pUC18 including EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, PstI, SphI and HindIII are unique in YEp352; these sites are also unique in YEp351 except for EcoRI and KpnI, which occur twice; (5) recombinant plasmids with DNA inserts in the multiple cloning region of YEp351 and YEp352 can be recognised by loss of beta-galactosidase function in appropriate E. coli hosts; (6) YEp351 and YEp352 contain the yeast LEU2 and URA3 genes, respectively, allowing for selection of these auxotrophic markers in yeast and E. coli; (7) both plasmids are retained with high frequency in yeast grown under non-selective conditions indicative of high plasmid copy number. The above properties make the shuttle vectors suitable for construction of yeast genomic libraries and for cloning of DNA fragments defined by a large number of different restriction sites. The two vectors have been further modified by deletion of the sequences necessary for autonomous replication in yeast. The derivative plasmids YIp351 and YIp352 can therefore be used to integrate specific sequences into yeast chromosomal DNA.

Toward a physical map of the genome of the nematode Caenorhabditis elegans

Abstract: A technique for digital characterization and comparison of DNA fragments, using restriction enzymes, is described. The technique is being applied to fragments from the nematode Caenorhabditis elegans (i) to facilitate cross-indexing of clones emanating from different laboratories and (ii) to construct a physical map of the genome. Eight hundred sixty clusters of clones, from 35 to 350 kilobases long and totaling about 60% of the genome, have been characterized.

Random-clone strategy for genomic restriction mapping in yeast.

Abstract: An approach to global restriction mapping is described that is applicable to any complex source DNA. By analyzing a single restriction digest for each member of a redundant set of lambda clones, a data base is constructed that contains fragment-size lists for all the clones. The clones are then grouped into subsets, each member of which is related to at least one other member by a significant overlap. Finally, a tree-searching algorithm seeks restriction maps that are consistent with the fragment-size lists for all the clones in each subset. The feasibility of the approach has been demonstrated by collecting data on 5000 lambda clones containing random 15-kilobase inserts of yeast DNA. It is shown that these data can be analyzed to produce regional maps of the yeast genome, extending in some cases for over 100 kilobases. In combination with hybridization probes to previously cloned genes, these local maps are already useful for defining the physical arrangement of closely linked genes. They may in the future serve as building blocks for the construction of a continuous global map.

DNA finger printing by oligonucleotide probes specific for simple repeats.

Abstract: Interspersed simple repetitive DNA is a convenient genetic marker for analysis of restriction fragment length polymorphisms (RFLPs) because of the numbers and the frequencies of its alleles. Oligonucleotide probes specific for variations of the GATCA simple repeats have been designed and hybridized to a panel of human DNAs digested with various restriction enzymes. Numerous RFLPs were demonstrated in AluI and MboI digested DNA with "pure" GATA oligonucleotides as probes. The optimal length of the probe for RFLP analysis was 20 bases taking into account fragment lengths (1.5-7 kilobases = kb), signal to background ratio, and number of clearly evaluable RFLPs. By using different restriction enzymes individual-specific hybridization patterns ("DNA fingerprints") can be established. Hypervariable simple repeat fragments are stably inherited in a Mendelian fashion. Advantages of this method are discussed.

Identification of a plasmid-borne parathion hydrolase gene from Flavobacterium sp. by southern hybridization with opd from Pseudomonas diminuta.

Abstract: Parathion hydrolases have been previously described for an American isolate of Pseudomonas diminuta and a Philippine isolate of Flavobacterium sp. (ATCC 27551). The gene which encodes the broad-spectrum organophosphate phosphotriesterase in P. diminuta has been shown by other investigators to be located on a 66-kilobase (kb) plasmid. The intact gene (opd, organophosphate-degrading gene) from this degradative plasmid was cloned into M13mp10 and found to express parathion hydrolase under control of the lac promoter in Escherichia coli. In Flavobacterium sp. strain ATCC 27551, a 43-kb plasmid was associated with the production of parathion hydrolase by curing experiments. The M13mp10-cloned fragment of the opd gene from P. diminuta was used to identify a homologous genetic region from Flavobacterium sp. strain ATCC 27551. Southern hybridization experiments demonstrated that a genetic region from the 43-kb Flavobacterium sp. plasmid possessed significant homology to the opd sequence. Similar hybridization did not occur with three other native Flavobacterium sp. plasmids (approximately 23, 27, and 51 kb) present within this strain or with genomic DNA from cured strains. Restriction mapping of various recombinant DNA molecules containing subcloned fragments of both opd plasmids revealed that the restriction maps of the two opd regions were similar, if not identical, for all restriction endonucleases tested thus far. In contrast, the restriction maps of the cloned plasmid sequences outside the opd regions were not similar. Thus, it appears that the two discrete bacterial plasmids from parathion-hydrolyzing soil bacteria possess a common but limited region of sequence homology within potentially nonhomologous plasmid structures.

Fine structure of the human hypoxanthine phosphoribosyltransferase gene

Abstract: The human hypoxanthine phosphoribosyltransferase (HPRT) gene has been characterized by molecular cloning, mapping, and DNA sequencing techniques. The entire gene, which is about 44 kilobases in length, is composed of nine exon elements. The positions of the introns within the coding sequence are identical to those of the previously-characterized mouse HPRT gene, although there are significant differences between intron sizes for the two genes. HPRT minigenes have been used in a transient expression assay involving microinjection into HPRT- cells to demonstrate functional promoter activity within a 234-base-pair region upstream from the ATG codon. The promoter of this gene resembles those of other recently characterized "housekeeping" genes in that it lacks CAAT- and TATA-like sequences, but contains several copies of the sequence GGGCGG. Both RNase protection and primer extension analysis indicate that human HPRT mRNA is heterogeneous at the 5' terminus, with transcription initiation occurring at sites located congruent to 104 to congruent to 169 base pairs upstream from the ATG codon. Comparison of the mouse and human HPRT 5'-flanking sequences indicates that there are only limited stretches of conserved sequence, although there are other shared features, such as an extremely high density of potential methylation sites, that may have functional significance.

Molecular population genetics of the alcohol dehydrogenase gene region of drosophila melanogaster

Abstract: Variation in the DNA restriction map of a 13-kb region of chromosome II including the alcohol dehydrogenase structural gene (Adh) was examined in Drosophila melanogaster from natural populations. Detailed analysis of 48 D. melanogaster lines representing four eastern United States populations revealed extensive DNA sequence variation due to base substitutions, insertions and deletions. Cloning of this region from several lines allowed characterization of length variation as due to unique sequence insertions or deletions [nine sizes; 21–200 base pairs (bp)] or transposable element insertions (several sizes, 340 bp to 10.2 kb, representing four different elements). Despite this extensive variation in sequences flanking the Adh gene, only one length polymorphism is clearly associated with altered Adh expression (a copia element approximately 250 bp 5' to the distal transcript start site). Nonetheless, the frequency spectra of transposable elements within and between Drosophila species suggests they are slightly deleterious. Strong nonrandom associations are observed among Adh region sequence variants, ADH allozyme (Fast vs. Slow), ADH enzyme activity and the chromosome inversion ln(2L) t. Phylogenetic analysis of restriction map haplotypes suggest that the major twofold component of ADH activity variation (high vs. low, typical of Fast and Slow allozymes, respectively) is due to sequence variation tightly linked to and possibly distinct from that underlying the allozyme difference. The patterns of nucleotide and haplotype variation for Fast and Slow allozyme lines are consistent with the recent increase in frequency and spread of the Fast haplotype associated with high ADH activity. These data emphasize the important role of evolutionary history and strong nonrandom associations among tightly linked sequence variation as determinants of the patterns of variation observed in natural populations.

Cloning and sequence analysis of cDNA for bovine carboxypeptidase E.

Abstract: Carboxypeptidase E (enkephalin convertase) was first identified as the carboxypeptidase B-like enzyme involved in the biosynthesis of enkephalin in bovine adrenal chromaffin granules1. A similar enzyme is present in many brain regions1,2 and in purified secretory granules from rat pituitary3 and rat insulinoma4. Within the secretory granules, carboxypeptidase E (CPE) activity is found in both a soluble and a membrane-bound form1, which differ slightly in relative molecular mass (Mr)5. Here, to investigate whether the CPE activities in the various tissues are produced from a single gene, purified CPE was partially sequenced and oligonucleotide probes were used to isolate a clone encoding CPE from a bovine pituitary complementary DNA library. This cDNA hybridizes to bovine pituitary poly(A)+ RNAs of approximately 3.3, 2.6 and 2.1 kilobases (kb), with the 3.3-kb messenger RNA the predominant species. The predicted amino-acid sequence of the cDNA clone contains the partially determined sequences of CPE, several pairs of basic amino acids and displays some homology with both carboxypeptidases A and B. Restriction analysis of bovine genomic DNA suggests only one gene for CPE. This is consistent with a broad role for CPE in the biosynthesis of many neuropeptides.

Long-range restriction site mapping of mammalian genomic DNA.

Abstract: Molecular analysis of many problems in genetics would be facilitated by the ability to construct restriction site maps of long stretches of genomic DNA and to directly place genes on these maps. Pulsed-field gradient gel electrophoresis allows measurement of the size of DNA fragments up to at least 2,000 kilobase pairs (kb) long and we have used this technique here to map sites for one class of infrequently cutting restriction enzyme over a total of 1,500 kb of mouse genomic DNA. The sites for these enzymes tend to be clustered in the genome. These clusters may correspond to the short stretches of C + G-rich unmethylated DNA often associated with mammalian genes.

Molecular structure and polymorphic map of the human phenylalanine hydroxylase gene.

Abstract: Human phenylalanine hydroxylase is a liver-specific enzyme that catalyzes the conversion of phenylalanine to tyrosine. Absence of enzymatic activity results in phenylketonuria, a genetic disorder that causes development of severe mental retardation in untreated children. In this paper we report the cloning and structure of the normal human phenylalanine hydroxylase gene, which was isolated in four overlapping cosmid clones that span more than 125 kilobases (kb) of the genetic locus. The peptide coding region of the gene is about 90 kb in length and contains 13 exons, with intron sizes ranging from 1 to 23 kb. Exons at the 3' half of the gene are compact, whereas those at the 5' half are separated by large introns. The human phenylalanine hydroxylase gene codes for a mature messenger RNA of approximately 2.4 kb, and its noncoding to coding DNA ratio is one of the highest among eukaryotic genes characterized to date. The map positions of nine polymorphic restriction sites identified within the locus were established by restriction enzyme mapping of the cloned gene fragments. Two clusters of polymorphic sites were demonstrated: (1) BglII, PvuII(a), and PvuII(b) at the 5' end of the gene and (2) EcoRI, XmnI, MspI(a), MspI(b), EcoRV, and HindIII at the 3' end. The polymorphic site distribution within this gene is a useful tool for prenatal diagnosis and carrier detection of the genetic disorder, while knowledge of normal gene structure is a prerequisite for future characterization of mutant alleles.

Glutathione reductase from Escherichia coli: cloning and sequence analysis of the gene and relationship to other flavoprotein disulfide oxidoreductases.

Abstract: A glutathione reductase negative strain of Escherichia coli K-12 was isolated as a thermoresistant survivor when a gor::MuctsAp lysogen was subjected to elevated temperature. It was found that in addition to being ampicillin sensitive this mutant was hypersensitive to arsenate, which may be connected with the fact that the gor gene maps between 77 and 78 min on the E. coli genome, close to the pit locus encoding the major arsenate transport system of E. coli. A derivative of this mutant was used as the recipient in a screen of the Clarke and Carbon hybrid plasmid bank of E. coli DNA. A plasmid, pGR, was isolated that encodes both an arsenate-resistance element and glutathione reductase. Restriction mapping of this plasmid showed that the insert DNA is approximately 10 kilobase pairs in length, and a fragment of the gor gene was identified that allowed the gor gene to be accurately mapped on pGR by a combination of restriction analysis and Southern blotting. The DNA sequence of the gor gene was determined and found to encode a protein of 450 amino acid residues. The glutathione reductase of E. coli is very homologous to the human enzyme and is also related (though less closely) to other flavoprotein disulfide oxidoreductases whose sequences are available. These enzymes have retained a common mechanism while evolving different specificities.

Somatic mutations at a heterozygous autosomal locus in human cells occur more frequently by allele loss than by intragenic structural alterations.

Abstract: A human B-cell lymphoblastoid cell line heterozygous at the thymidine kinase (TK)locus (i.e., carrying one functional and one nonfunctional thymidine kinase allele) was used to study the molecular nature of mutations leading to loss of TKactivity. A total of 113 mutant clones, both spontaneous and induced, were examined by restriction enzyme mapping and by the use of a restriction fragment length polymorphism (RFLP) at the TKlocus. A majority (71%) of all mutant clones examined had lost the entire functional TKallele, becoming either homozygous or hemizygous for the nonfunctional allele. The remaining mutants had either no detectable changes (26%) or had obvious structural alterations (less than 5%) in the active TKgene. These results emphasize the importance of allele loss, presumably by mitotic chromosomal mechanisms, in mutagenesis at autosomal loci, and suggest that in vitro models for recessive somatic mutation which are based at hemizygous loci may ignore a large category of genetically significant events.

Sequence analysis of the major outer membrane protein gene from Chlamydia trachomatis serovar L2.

Abstract: The structural gene for the major outer membrane protein (MOMP) from Chlamydia trachomatis was cloned and sequenced A lambda gt11 recombinant (lambda gt11/L2/33) that contains a portion of the MOMP coding sequence was used to probe a lambda 1059 library constructed from DNA obtained from C trachomatis serovar L2 Selected lambda 1059 recombinants were mapped with endonuclease restriction enzymes The MOMP gene was mapped to the 5' end of a BamHI fragment of approximately 9 kilobases Contiguous endonuclease restriction fragments identified within this region permitted the selection of specific fragments for subcloning and DNA sequencing The MOMP gene consisted of a 1,182-base-pair open reading frame that encoded 394 amino acids and ended with three stop codons The known amino-terminal amino acid was preceded by 22 amino acids whose sequence was compatible with a leader or signal sequence The primary structure of MOMP determined from the translated DNA sequence demonstrated nine cysteine residues and a remarkably homogeneous distribution of charged and hydrophobic residues

Charomids: cosmid vectors for efficient cloning and mapping of large or small restriction fragments.

Abstract: Charomids are cosmid vectors up to 52 kilobases (kb) long, bearing 1-23 copies of a 2-kb spacer fragment linked in head-to-tail tandem arrays. Like cosmids and lambda phage, charomids can be packaged in vitro for efficient introduction into bacteria. Charomids contain a polylinker with nine unique restriction sites for cloning and can be used without preparing vector arms. Using a charomid of appropriate size, one can clone inserts of any size up to 45 kb. For example, charomid 9-36 (9 cloning sites, 36 kb long) is too small to be packaged efficiently without an insert and can be used to clone fragments of 2-16 kb. The structure of charomids facilitates restriction mapping of the insert DNA and, after cloning, all the spacer fragments can be removed easily. After enrichment by size fractionation in an agarose gel, a specific single-copy genomic sequence can be cloned rapidly from approximately 3 micrograms of DNA. Using charomid 9-36, we have cloned and mapped an amplified novel DNA fragment from a cell line resistant to N-(phosphonoacetyl)-L-aspartate and carrying about 100 copies of the CAD (carbamoyl-phosphate synthetase/aspartate carbamoyltransferase/dihydroorotase) gene. The fragment lies at the center of an inverted duplication of this gene.

The organization of the human HPRT gene

Abstract: The organization of the X-linked gene for human hypoxanthine phosphoribosyltransferase (HPRT, EC 2.4.2.8.) has been determined by a combination of restriction endonuclease mapping, heteroduplex analysis and DNA sequence analysis of overlapping genomic clones. The entire gene is 42 kilobases in length and split into 9 exons. The sizes of the 7 internal exons and the exon-intron boundaries are identical to those of mouse HPRT gene. The 5' end of the gene lacks the prototypical 5' transcriptional regulatory sequence elements but contains extremely GC-rich sequences and five GC hexanucleotide motifs (5'-GGCGGG-3'). These structural features are very similar to those found in the mouse HPRT gene and to some of the regulatory signals common to a class of constitutively expressed "housekeeping" genes. Several transcriptional start sites have been identified by nuclease protection studies. Extensive sequence homology between the mouse and human genes is found in the 3' non-coding portion of the gene.

Cloning and expression of the pilin gene of Pseudomonas aeruginosa PAK in Escherichia coli.

Abstract: Many strains of Pseudomonas aeruginosa possess pili which have been implicated in the pathogenesis of the organism. This report presents the cloning and expression in Escherichia coli of the gene encoding the structural subunit of the pili of P. aeruginosa PAK. Total DNA from this strain was partially digested with Sau3A and inserted into the cloning vector pUC18. Recombinant E. coli clones were screened with oligonucleotide probes prepared from the constant region of the previously published amino acid sequence of the mature pilin subunit. Several positive clones were identified, and restriction maps were generated. Each clone contained an identical 1.1-kilobase HindIII fragment which hybridized to the oligonucleotide probes. Western blot analysis showed that all of the clones expressed small amounts of the P. aeruginosa pilin subunit, which has a molecular mass of ca. 18,000. This expression occurred independently of the orientation of the inserted DNA fragments in the cloning vector, indicating that synthesis was directed from an internal promoter. However, subclones containing the 1.1-kilobase HindIII fragment in a specific orientation produced an order of magnitude more of the pilin subunit. While the expressed pilin antigen was located in both the cytoplasmic and outer membrane fractions of E. coli, none appeared to be polymerized into a pilus structure.

Immunoglobulin heavy chain locus of the rat: striking homology to mouse antibody genes.

Abstract: DNA encoding the rat diversity segment (D), joining segment (JH), and constant (C) region mu, gamma 2a, gamma 1, gamma 2b, epsilon and alpha of the Ig heavy chain has been isolated from a cosmid library. Restriction mapping allowed us to identify two gene clusters: D-JH-C mu and C gamma 1-C gamma 2b-C epsilon-C alpha in addition to a single C gamma 2a gene. Analysis of genomic DNA by Southern blotting permitted identification of the C gamma 2c gene and led to the proposal of the following gene order for the rat Ig heavy chain locus: D-JH-C mu-C delta-(C gamma 2c, C gamma 2a)-C gamma 1-C gamma 2b-C epsilon-C alpha. There is striking homology between the rat and mouse Ig heavy chain loci as regards gene order and distance between CH genes. Partial DNA sequencing confirms this homology and shows that exon sequences are more conserved than are intron sequences. One of the most conserved intron regions between rat and mouse is that spanning the Ig heavy chain enhancer (91% homology). However, the relationship between the different C gamma subclasses in rat differs from that in mouse. Comparison of the C gamma CH3 domains shows that the rat C gamma 2b gene is most homologous to mouse C gamma 2a/b, whereas the rat C gamma 1 and C gamma 2a genes, both very similar to each other, are most homologous to the mouse C gamma 1 gene.

Isolation and characterization of the two structural genes coding for phosphofructokinase in yeast.

Abstract: Yeast phosphofructokinase is an octamer composed of two different kinds of subunit. The genes coding for these subunits have been isolated by means of functional complementation in a pfk1 pfk2 double mutant. As a source of DNA the genomic library of Nasmyth and Tatchell (1980) constructed in the yeast multicopy vector YEp13 was used. Plasmids containing the information of one or the other gene were identified by back-transformation into pfk single mutants (pfk1 PFK2, PFK1 pfk2). Restriction maps of the respective insertions are provided. The genomic organization was confirmed by Southern analysis. Northern analysis showed hybridization to mRNAs of about 3.6 kb for both genes, corresponding to the molecular weight of the protein subunits. Transformation with one of the plasmids did not lead to an increase in phosphofructokinase activity. Subcloning of both genes in one multicopy vector (YEp13) and reintroduction into the yeast cell resulted in a 3.5-fold higher specific activity compared to the wild type. Overproduction of the protein subunits in this transformant was confirmed by SDS-polyacrylamide electrophoresis of crude extracts stained with Coomassie-blue. This was not accompanied by an increased ethanol production. The sequences encoding the two subunits were shown to share homology.

Expression in Escherichia coli and function of Pseudomonas aeruginosa outer membrane porin protein F.

Abstract: The gene encoding porin protein F of Pseudomonas aeruginosa was cloned onto a cosmid vector into Escherichia coli. Protein F was expressed as the predominant outer membrane protein in a porin-deficient E. coli background and was clearly visible on one-dimensional sodium dodecyl sulfate-polyacrylamide gels in a porin-sufficient background. The identity of the protein F from the E. coli clone and native P. aeruginosa protein F was demonstrated by their identical mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoretograms, 2-mercaptoethanol modifiabilities, and reactivities with monoclonal antibodies specific of two separate epitopes of protein F. In the course of gene subcloning, a 2-kilobase DNA fragment was isolated, with an apparent truncation of the part of the gene encoding the carboxy terminus of protein F. This subclone produced a 24,000-molecular-weight, outer membrane-associated, truncated protein F derivative which was not 2-mercaptoethanol modifiable and which reacted with only one of the two classes of protein F-specific monoclonal antibodies. The 2-kilobase fragment was used in Southern blot hybridizations to construct a restriction map of the cloned and subcloned fragments and to demonstrate with restriction digests of whole P. aeruginosa DNA that only one copy of the protein F gene was present in the P. aeruginosa chromosome. The protein F produced by the original cosmid clone in a porin-deficient E. coli background was purified. To demonstrate retention of porin function after cloning, the protein F from the E. coli clone was incorporated into black lipid bilayer membranes. Two major classes of channels were revealed. The predominant class of channels had an average conductance of 0.36 nS in 1 M KCl, whereas larger channels (4 to 7 nS) were seen at a lower frequency. Similar channel sizes were observed for porin protein F purified by the same method from P. aeruginosa outer membranes. Images

Early postimplantation embryo lethality due to DNA rearrangements in a transgenic mouse strain.

Abstract: Insertional mutagenesis in a transgenic mouse strain (HUGH/3) was caused by integration of plasmid DNA containing the human growth hormone gene and pBR322 plasmid sequences. From this study, which includes another instance of mutagenesis, and from other reports, it is apparent that insertional mutagenesis occurs fairly frequently during DNA integration in the mouse egg and that it is not specific for the exogenous DNA employed. The mutation in HUGH/3 is recessive and results in death of embryos homozygous for the donor sequences shortly after implantation, at the egg cylinder stage on days 4-5 of gestation. Restriction mapping of the insert and of the flanking DNA regions indicates that integration must have involved a series of complex events. Approximately five copies of plasmid sequences are arrayed in tandem but are interrupted at least twice by mouse cellular sequences. In addition, the mouse flanking DNA shows extensive rearrangements, probably including a deletion of at least 10 kilobases. The rearrangements may reflect an initially unstable DNA structure followed by attainment of a more stable conformation.

Structure of the highly repeated, long interspersed DNA family (LINE or L1Rn) of the rat.

Abstract: We present the DNA sequence of a 6.7-kilobase member of the rat long interspersed repeated DNA family (LINE or L1Rn). This member (LINE 3) is flanked by a perfect 14-base-pair (bp) direct repeat and is a full-length, or close-to-full-length, member of this family. LINE 3 contains an approximately 100-bp A-rich right end, a number of long (greater than 400-bp) open reading frames, and a ca. 200-bp G + C-rich (ca. 60%) cluster near each terminus. Comparison of the LINE 3 sequence with the sequence of about one-half of another member, which we also present, as well as restriction enzyme analysis of the genomic copies of this family, indicates that in length and overall structure LINE 3 is quite typical of the 40,000 or so other genomic members of this family which would account for as much as 10% of the rat genome. Therefore, the rat LINE family is relatively homogeneous, which contrasts with the heterogeneous LINE families in primates and mice. Transcripts corresponding to the entire LINE sequence are abundant in the nuclear RNA of rat liver. The characteristics of the rat LINE family are discussed with respect to the possible function and evolution of this family of DNA sequences.

Location and Nucleotide Sequence of the Orgyia pseudotsugata Single Nucleocapsid Nuclear Polyhedrosis Virus Polyhedrin Gene

Abstract: A restriction endonuclease map was determined for the Orgyia pseudotsugata single nucleocapsid nuclear polyhedrosis virus (SNPV) genome. The order of the fragments generated by the enzymes BglII, BamHI and XbaI was analysed using double digestion of the total genome and digestion of isolated restriction fragments. The location of the polyhedrin gene was then determined using a cloned polyhedrin gene from the O. pseudotsugata multiple nucleocapsid NPV (MNPV) as a hybridization probe. A fragment containing this gene was cloned, mapped, subcloned and the nucleotide sequence of a 1.3 kb fragment was determined which contained the entire polyhedrin reading frame and some flanking sequences. This gene demonstrated 76% nucleotide sequence homology and 87% amino acid sequence homology to the Autographa californica MNPV polyhedrin sequence. A probable regulatory element was identified which is common to the 5' flanking region of all hypertranscribed late genes (polyhedrin and 10K proteins) which have been examined in baculoviruses.