BamHI Restriction Fragment Research Articles

Rolling circle amplification of begomoviral DNA from a single nucleus isolated by laser dissection microscopy

Laser dissection microscopy (LDM) is a method for isolating organelles, a specific cell or cells/tissue of interest from microscopic regions with the help of a laser. Here we describe a LDM-based isolation of begomovirus infected Nicotiana benthamiana epidermal cells and nuclei, in combination with a fast method to prepare non-fixed leaf epidermal samples for LDM. The bipartite Abutilon mosaic virus (AbMV) was used in which the coat protein gene of DNA A was deleted and replaced by the open reading frame (ORF) coding for the green fluorescent protein (GFP, accession: U87624), agro-infiltrated together with DNA B, to visualize infected cells. GFP expressing epidermal cells or nuclei were isolated by LDM with the MMi Cellcut system and viral circular DNA was amplified by rolling circle amplification (RCA). Subsequently, the RCA product was incubated with the restriction enzymes BamHI and PstI and restriction fragments were separated on an agarose gel to prove presence of the viral genome. It was shown that even a single-isolated nucleus harbored enough material to produce a sufficient restriction fragment pattern to identify a begomovirus infected cell/nucleus.

Differentiation of Clavibacter michiganensis subsp. sepedonicus using PCR melting profile and variable number of tandem repeat methods.

The potato phytopathogen Clavibacter michiganensis subsp. sepedonicus (Cms) is a causative agent of bacterial ring rot, which is a serious threat to crops. In EU member countries, Cms is subject to quarantine and has to be combated. The knowledge about the transmission of C. michiganensis strains is limited due to a lack of methods which could be used for epidemiological analysis. In this study, PCR melting profile (PCR MP) and variable number tandem repeat methods were used in Cms epidemiological analysis for the first time. PCR MP was based on the melting temperature analysis of BamHI restriction fragments of chromosomal DNA. Respectively, for the variable number tandem repeat (VNTR) method, six loci were identified and used in the differentiation of Cms isolates. PCR MP was used for 93 Cms isolated in Poland. Both PCR MP and VNTR methods were used for the differentiation of 47 Cms strains in this collection. Both these methods were found to be useful for the epidemiological analysis of Cms. SIGNIFICANCE AND IMPACT OF THE STUDY: The potato phytopathogen, Clavibacter michiganensis subsp. sepedonicus (Cms), is a serious threat to crops and lead to significant economic losses. The only way to control and eliminate the disease caused by this pathogen is the use of certified seed potato and strict quarantine of infected fields. Here, for the first time, two molecular typing methods (PCR melting profile (PCR MP) and variable number tandem repeat (VNTR)) were evaluated in respect of their potential in differentiation of Cms isolates. As a result, we obtained characteristic profiles of DNA fragments (PCR MP) and numeric patterns (VNTR), which enable the intraspecies genotyping of Cms strains confirming the effectiveness of PCR MP and VNTR methods in differentiation of Cms strains.

Isolation and characterization of pseudorabies virus from a wolf (Canis lupus) from Belgium

Aujeszky’s disease is an economically important disease in domestic pigs caused by the alphaherpesvirus pseudorabies virus (PRV). However, also wild boars are a natural reservoir for the virus, and this can lead to infection of wildlife carnivore species. Three wolves held in the wildlife park of Han-sur-Lesse in the province of Namur in Belgium were suspected to be infected with PRV based on the nervous symptoms they showed after being fed with wild boar offal. The diagnosis was confirmed for a female wolf by a positive real-time PCR detecting PRV. The virus was isolated from the brain tissue of the wolf and characterized by restriction fragment length polymorphism analysis and phylogenetic analysis. The obtained BamHI restriction fragment pattern of the wolf isolate was similar to that of the reference strain Kaplan, thereby characterizing it as a type Ip isolate. Type I PRV strains, and particularly subtype Ip, are predominant in European wild boar. Phylogenetic analysis based on the sequence of a fragment of glycoprotein C showed that the Belgian isolate belonged to cluster B and that the sequence was identical to that of wild boar isolates from southwestern Germany, eastern France, and Spain. This study is the first report of Aujeszky’s disease in wolves and shows that they are susceptible to PRV by eating infected wild boar offal leading to fatal neurological disease. This illustrates the possible implications of PRV-infected wild boar for the conservation of wolves and other carnivore species.

Molecular characterization and genetic organization of the inhibitor of apoptosis gene (iap-5) region of the Pieris rapae granulovirus

Pieris rapae granulovirus (PiraGV) is a baculovirus pathogenic to the insect P. rapae (Pieridae, Lepidoptera). Though being known for decades, information on the genetic organization of this virus remains limited. In an effort to characterize this virus, an 11.8 kb BamHI restriction fragment that harbors the inhibitor of apoptosis gene (iap-5) was sequenced. Our results indicate that this region contains important genes such as dnapol, lef-3, lef-9, and dnaligase that are involved in transcription and replication of the virus. The gene content and synteny in this region are highly conserved among granulovirus genomes. Phylogenetic analysis showed that PiraGV genes are more closely related to the Choristoneura occidentalis granulovirus (ChocGV) than other characterized granulovirus (GVs).

Association of gene polymorphisms for plasminogen activators with alveolar bone loss

The plasminogen activating system is a protease/inhibitor system central to extracellular matrix remodeling with a suggested role in periodontal disease pathology. A few studies have reported polymorphisms in the genes of plasminogen activator inhibitors to be associated with periodontal disease severity. Two gene polymorphisms - a BamHI restriction fragment length polymorphism in the urokinase plasminogen activator gene (uPA) and a HindIII restriction fragment length polymorphism in the plasminogen activator inhibitor type 1 gene (PAI-1) - have been associated with conditions having a vascular component, and our objective was to assess the association of these gene polymorphisms with alveolar bone loss in chronic periodontal disease of adults. Genotype was determined by polymerase chain reaction amplification of whole blood, pertinent histories were obtained by interview, and alveolar bone loss was assessed from current radiographs. In 77 elderly patients with a normal distribution of alveolar bone loss, we demonstrated a significant association between levels of alveolar bone loss and these polymorphisms in the uPA and PAI-1 genes. Controlling for the contributions of smoking or diabetes to periodontal bone loss, estimated odds ratios for predicting lower levels of alveolar bone loss, associated with a greater degree of periodontal health, were strongest when defined by the concurrent presence of a homozygous urokinase plasminogen activator genotype and the nuclease-sensitive plasminogen activator inhibitor type 1 (HindIII) allele (odds ratio = 2.6; 95% confidence interval: 5.8-1.3). The urokinase plasminogen activator (BamHI) and plasminogen activator inhibitor type 1 (HindIII) genotypes may serve as useful markers for heritability of bone loss associated with periodontal disease.

Organization of the interferon-inducible 2',5'-oligoadenylate-dependent ribonuclease L (RNase L) gene of mouse.

Interferons (IFNs) induce a 2',5'-oligoadenylate (2-5A)-dependent ribonuclease L (RNase L) following virus-infection of mammalian cells. RNase L degrades both viral and cellular RNAs and restricts virus-proliferation. We have studied organization of RNase L gene in genomic DNA from the mouse liver by Southern blot analysis. Several BamHI, BglII, EcoRI, HincII, HindIII, NcoI, PstI, SacI, and XbaI restriction fragments hybridized to (32)P-labeled mouse RNase L cDNA and the 5'-proximal exon probes. Mouse RNase L gene exists as a single copy (>16 kb DNA) gene. A 5 kb HindIII and a 2.5 kb EcoRI DNA were detected as 5'-upstream DNA of the gene which may contain mouse RNase L promoter. Our results will help studying mouse RNase L gene promoter in further details.

Cloning, sequencing and application of the LEU2 gene from the sour dough yeast Candida milleri

We have cloned by complementation in Saccharomyces cerevisiae and sequenced a LEU2 gene from the sour dough yeast Candida milleri CBS 8195 and studied its chromosomal location. The LEU2 coding sequence was 1092 nt long encoding a putative beta-isopropylmalate dehydrogenase protein of 363 amino acids. The nucleotide sequence in the coding region had 71.6% identity to S. cerevisiae LEU2 sequence. On the protein level, the identity of C. milleri Leu2p to S. cerevisiae Leu2p was 84.1%. The CmLEU2 DNA probe hybridized to one to three chromosomal bands and two or three BamHI restriction fragments in C. milleri but did not give any signal to chromosomes or restriction fragments of C. albicans, S. cerevisiae, S. exiguus or Torulaspora delbrueckii. Using CmLEU2 probe for DNA hybridization makes it easy to quickly identify C. milleri among other sour dough yeasts.

Identification, Characterization and Phylogenic Analysis of Conserved Genes within the p74 Gene Region of Choristoneura fumiferana Granulovirus Genome

The genes located within the p74 gene region of the Choristoneura fumiferana granulovirus (ChfuGV) were identified by sequencing an 8.9 kb BamHI restriction fragment on the ChfuGV genome. The global guanine-cytosine (GC) content of this region of the genome was 33.02%. This paper presents the ORFs within the p74 gene region along with their transcriptional orientations. This region contains a total of 15 open reading frames (ORFs). Among those, 8 ORFs were found to be homologues to the baculoviral ORFs: Cf-i-p , Cf-vi, Cf-vii, Cf-viii (ubiquitin), Cf-xi (pp31), Cf-xii (lef-11), Cf-xiii (sod) and Cf-xv-p (p74). To date, no specific function has been assigned to the ORFs: Cf-i, Cf-ii, Cf-iii, Cf-iv, Cf-v, Cf-vi, Cf-vii, Cf-ix and Cf-x. The most noticeable ORFs located in this region of the ChfuGV genome were ubiquitin, lef-11, sod, fibrillin and p74. The phylogenetic trees (constructed using conceptual products of major conserved ORFs) and gene arrangement in this region were used to further examine the classification of the members of the granulovirus genus. Comparative studies demonstrated that ChfuGV along with the Cydia pomonella granulovirus (CpGV), Phthorimaea operculella granulovirus (PhopGV), Adoxophyes orana granulovirus (AoGV) and Cryptophlebia leucotreta granulovirus (ClGV) share a high degree of amino acids sequence and gene arrangement preservation within the studied region. These results support a previous report, which classified a granuloviruses into 2 distinct groups: Group I: ChfuGV, CpGV, PhopGV and AoGV and Group II: Xestia c-nigrum granulovirus (XcGV) and Plutella xylostella granulovirus (PxGV). The phylogenetic and gene arrangement studies also placed ClGV as a novel member of the Group I granuloviruses.

Single Nucleotide Polymorphism analysis of the human adenosine A2B receptor gene: prevalence of SNPs in asthmatics and normal subjects

AbstractThe adenosine A2B receptor is found on human lung mast cells and is believed to mediate the bronchoconstriction in response to adenosine characteristic of asthmatics. As such it represents an attractive therapeutic target for asthma and allergic rhinitis. As genetic variability in drug targets may affect an individual's response to treatment, the adenosine A2B receptor gene was analyzed for polymorphisms and their prevalence defined in a Caucasian population. The coding region of the A2B receptor gene as well as the intron‐exon boundaries of the gene were found to be free of genetic variation. Three single nucleotide polymorphisms were identified in the promoter region of the gene, one of which created a BamHI restriction fragment length polymorphism. The prevalence of the three single nucleotide polymorphisms in an unselected population was <5%. The prevalence of the BamHI polymorphism was investigated in a British population of 40 asthmatics and 40 non‐asthmatics: no excess of the BamHI variant was observed in asthmatic subjects. The data indicates that any variability in drug response would likely not be due to direct structural variation of the A2B receptor protein. Further, polymorphism at the A2B receptor genomic locus is unlikely to contribute to the population risk of developing asthma.

Aflatoxin genes and the aflatoxigenic potential of Koji moulds.

Sixty-four Aspergillus isolates, 54 of which originated from food fermentations, and 18 Aspergillus reference strains were identified and screened for the presence of aflatoxin genes aflR and omt-1. Among the Koji moulds, not only A. oryzae but also A. flavus strains were found. Furthermore, 27% of A. oryzae and 93% of A. flavus strains lacked either aflR or both aflR- and omt-1. A selection of 29 strains was also checked for the presence of pksA and nor-1. This revealed large deletions in the aflatoxin gene cluster of some strains. The hybridisation patterns also suggested a polarity in the deletion events, originating in the vicinity of pksA and extending towards omt-1. Other strains exhibited BamHI restriction fragment length polymorphisms (RFLPs) for either aflR or for aflR and omt-1. All aflR and/or omt-1 deletion strains turned out to be unable to produce aflatoxin. The RFLP-carrying strains either produced only traces of aflatoxin or none at all. In 73% of the A. oryzae strains, no apparent deletions were detected with the aflR and omt-1 probes. Nevertheless, after incubation in aflatoxin-inducing media, no aflatoxin B1 production could be detected in those A. oryzae strains.

Analysis of the Thiocapsa pfennigii polyhydroxyalkanoate synthase: subcloning, molecular characterization and generation of hybrid synthases with the corresponding Chromatium vinosum enzyme.

The PHA synthase structural gene of Thiocapsa pfennigii was identified and subcloned on a 2.8-kbp BamHI restriction fragment, which was cloned recently from a genomic 15.6-kbp EcoRI restriction fragment. Nucleotide sequence analysis of this fragment revealed three open reading frames (ORFs), representing coding regions. Two ORFs encoded for the PhaE (Mr 40,950) and PhaC (Mr 40,190) subunits of the PHA synthase from T. pfennigii and exhibited high homology with the corresponding proteins of the Chromatium vinosum (52.8% and 85.2% amino acid identity) and the Thiocystis violacea (52.5% and 82.4%) PHA synthases, respectively. This confirmed that the T. pfennigii PHA synthase was composed of two different subunits. Also, with respect to the molecular organization of phaE and phaC, this region of the T. pfennigii genome resembled very much the corresponding regions of C. vinosum and of Thiocystis violacea. A recombinant strain of Pseudomonas putida, which overexpressed phaE and phaC from T. pfennigii, was used to isolate the PHA synthase by a two-step procedure including chromatography on Procion Blue H-ERD and hydroxyapatite. The isolated PHA synthase consisted of two proteins exhibiting the molecular weights predicted for PhaE and PhaC. Hybrid PHA synthases composed of PhaE from T. pfennigii and PhaC from C. vinosum and vice versa were constructed and functionally expressed in a PHA-negative mutant of P. putida; and the resulting PHAs were analyzed.

Physical and genetic map of the Wiseana nucleopolyhedrovirus genome.

Wiseana nucleopolyhedrovirus (NPV) is the major pathogen of the New Zealand endemic pasture pest, Wiseana spp. To characterize this potential biological control agent, the genome of a virus isolated from Wiseana signata was purified and cloned. The complete genome was cloned as BamHI or HindIII restriction fragments, which were mapped by Southern hybridization and restriction analysis. To verify the physical map, the junctions between all HindIII fragments were confirmed by sequencing. The viral genome was estimated to be 128 kbp. Sequence data generated at the termini of cloned restriction fragments were compared to sequence databases to identify putative gene homologues. Seventeen putative ORFs, which were homologous to other baculoviral sequences, were identified. These putative ORFs were located on the Wiseana NPV physical map and their distribution was compared to genetic maps of NPVs isolated from Autographa californica, Orgyia pseudotsugata and Lymantria dispar. Although the virus from W. signata was significantly different from these other NPVs, a core region of the viral genome was conserved.

FHIT Gene Abnormalities in Both Benign and Malignant Thyroid Tumours

FHIT, a candidate tumour suppressor gene, has recently been identified at chromosomal region 3p14.2, and deletions of the gene have been reported in many types of human cancers. Loss of heterozygosity (LOH) at this region has also been found frequently in follicular thyroid carcinoma (FTC). To investigate the potential role of FHIT in thyroid tumorigenesis, we examined 57 thyroid tumour specimens (eight benign adenomas, 40 papillary, four follicular and five anaplastic carcinomas), and two thyroid carcinoma cell lines (NPA, SW579) for genetic alterations by using reverse transcription–polymerase chain reaction (RT–PCR), PCR product sequencing, single-strand conformation polymorphism (SSCP) and Southern blot analysis. Two cervical carcinoma cell lines (C-33A, HeLa) were included as positive controls. We detected truncated FHIT transcripts in three of eight (38%) benign adenomas, nine of 40 (23%) papillary, and two of five (40%) anaplastic carcinomas, and in three cell lines (SW579, C-33A, HeLa). Most of the truncated transcripts lacked exons 4 or 5 to 7 or 8 of the gene and were presumably non-functional as the translation start site is located in exon 5. SSCP analysis of the coding exons failed to detect any point mutations among the samples without abnormal FHIT transcripts. Southern blot analysis demonstrated either loss or reduced intensity of major Bam HI restriction fragments in the three cell lines found to have abnormal FHIT transcripts, indicating, respectively, either intragenic homozygous or heterozygous deletions of the FHIT gene. Intragenic homozygous deletions were also found in two papillary thyroid carcinoma specimens: one was missing a 13 kb Bam HI fragment which contains exon 4, the other had deletions of 15.5, 13 and 4.2 kb fragments which contain exons 2 and 9, 4, and 5, respectively. The absence of a defective FHIT gene in FTC indicates that an additional tumour suppressor gene may reside in this region and be involved in the development of FTC. Given that defective FHIT genes were found in both benign and malignant thyroid tumours, the inactivation of this putative tumour suppressor gene is likely to be an early event in the pathogenesis of some forms of thyroid neoplasms.

Lack of Association Between the Heparan Sulfate Proteoglycan Gene Polymorphism and Diabetic Nephropathy in Japanese NIDDM with Proliferative Diabetic Retinopathy

The development of diabetic nephropathy shows remarkable variation among individuals. Therefore, not only hvperglycemia hut also genetic factors may contribute to the development of diabetic nephropathy. Heparan sulfate proteoglycan (HSPG) is thought to play an important role as a component of the charge selectivity barrier in the glomerular basement membrane. Recently, a BamHI restriction fragment length polymorphism (RFLP) in the HSPG gene (HSPG 2) was reported to be associated with diabetic nephropathy in Caucasian insulin-dependent diabetes niellitus (IDDM). The aim of the present study was to examine the contribution of the BamHI HSPG2 polymorphism to the development of diabetic nephropathy in Japanese non-insulin-dependent diabetes mellitus (NIDDM). For this purpose, we recruited 102 patients with diabetic nephropathy and 64 age-matched patients without diabetic nephropathy from Japanese NIDDM patients. Since all the subjects had proliferative diabetic retinopathy, it seems likely that they would be exposed to hyperglycemia for a long time. In the present study, the BamHI HSPG2 genotype and allele frequencies were not significantly different between the patients with nephropathy and the patients without nephropathy. Therefore, we conclude that the BamHI HSPG2 polymorphism is not associated with the development of diabetic nephropathy in Japanese NIDDM.

Organisation of the equine immunoglobulin constant heavy chain genes: II. Equine c γ genes

The number of immunoglobulin G constant heavy chain genes ( cγ genes) varies broadly among mammalian species, reflecting structural and functional differences between expressed immunoglobulin G (IgG) isotypes and allotypes. Up to now equine IgG isotypes have been defined only at the biochemical and serological level. It is still not clear how many IgG isotypes exist in horses and whether there are any allotypes. Here, we describe the isolation and characterisation of equine cγ genes. An equine genomic lambda phage library was screened with a human c γ4 probe. Cross-hybridising equine c γ sequences were cloned twice and characterised by restriction mapping with the human c γ4 and a murine s γ1 probe. Genomic equine DNA probes for both, cγ genes and corresponding switch regions (s γ), were isolated and used for a more detailed BamHI restriction analysis, comparing genomic DNA of various horses. This analysis reveals the existence of at least five, or probably six cγ genes in the equine haploid genome. Beside the porcine system, this is the highest number of cγ genes described for any mammalian species. Moreover, for two of these cγ genes, BamHI restriction fragment length polymorphism became evident.

Identification of a BamHI Polymorphism for the Urokinase Gene Associated with Symptomatic Coronary Artery Disease.

Urokinase-type plasminogen activator (u-PA) and plasmin have been implicated in a number of processes, including activation of a variety of metalloproteinases, matrix remodeling, and cell migration, which may underlie the early initiation and progression of atherosclerosis and coronary artery disease (CAD). These studies were carried out to determine whether variations in the u-PA gene, using a BamHI restriction fragment length polymorphism (RFLP) as a new marker for genetic variation, may be associated with CAD. Southern blot analysis of individual digested genomic DNA (BamHI), hybridized with a 2-kb human u-PA cDNA probe, identified a two-allele RFLP with allelic bands at 6 and 1.5 kb. A constant band at 9 kb was detected. Three genotypes were identified and designated as 1/1 (6.0-kb band only), 1/2 (6.0 and 1.5-kb bands), and 2/2(1.5-kb band only). For these studies, 43, individual human umbilical cord samples, representing a "control" population, were analyzed and compared in terms of their u-PA genotypes with 34 saphenous vein samples from patients requiring coronary artery bypass grafting (CABG). Controls, presumed to reflect the normal population distribution, showed a u-PA genotype distribution of 1/1(n = 8, 18.6%), 1/2 (n = 33, 76.7%) and 2/2 (n = 2, 4.7%), whereas CAD patients showed a distribution of 1/1 (n = 16, 47.1%), 1/2 (n = 13, 38.2%), and 2/2 (n = 5, 14.7%). Comparison of the "control" genotype distribution with data derived from CABG patients demonstrated a significant difference in the distribution of u-PA genotypes (P = 0.002), with an increased prevalence of the homozygous 1/1 and 2/2 genotypes in CAD patients. These early studies demonstrate a significant association between u-PA gene polymorphism and the presence or absence of CAD.

Organization of the equine immunoglobulin constant heavy chain genes I. cε and cα genes

We provide a restriction map of the equine cε and cα genes as a molecular basis for isotype classification. Human and murine DNA probes were used for identification of homologous equine DNA sequences and for isolation of the equine cε and cα genes from a genomic DNA library. A detailed map of the equine 5′-sε/cε-sα/cα-3′ gene region was obtained. Equine cε and cα DNA probes were prepared and used for restriction analysis of immunoglobulin heavy chain gene loci from different horses. This analysis indicated the presence of only one equine cε and one cα gene in the haploid equine genome. In addition, for the equine cα gene, four haplotypes were identified according to BamHI restriction fragment length polymorphism (RFLP) of genomic DNA. The relative location of the cε and cα genes 3′ of the equine cμ and cγ genes was determined by restriction analysis of equi-murine heterohybridomas.

Characterization of Thinopyrum distichum chromosomes using double fluorescence in situ hybridization, RFLP analysis of 5S and 26S rRNA, and C-banding of parents and addition lines

Diagnostic markers for eight Thinopyrum distichum addition chromosomes in Triticum turgidum were established using C-banding, in situ hybridization, and restriction fragment length polymorphism analysis. The C-band karyotype conclusively identified individual Th. distichum chromosomes and distinguished them from chromosomes of T. turgidum. Also, TaqI and BamHI restriction fragments containing 5S and 18S-5.8S-26S rRNA sequences were identified as positive markers specific to Th. distichum chromosomes. Simultaneous fluorescence in situ hybridization showed both 5S and 18S-5.8S-26S ribosomal RNA genes to be located on chromosome IV. Thinopyrum distichum chromosome VII carried only a 18S-5.8S-26S rRNA locus and chromosome pair II carried only a 5S rRNA locus. The arrangement of these loci on Th. distichum chromosome IV was different from that on wheat chromosome pair 1B. Two other unidentified Th. distichum chromosome pairs also carried 5S rRNA loci. The homoeologous relationship between Th. distichum chromosomes IV and VII and chromosomes of other members of the Triticeae was discussed by comparing results obtained using these physical and molecular markers.

Plastid DNA of five ecotypes of Arabidopsis thaliana: sequence of ndhG gene and maternal inheritance

Southern blots of BamHI, HindIII and EcoRI restriction fragments with 21 heterologous and homologous probes indicated that restriction and gene maps of plastid DNA of Arabidopsis thaliana (L.) cv. Landsberg (L er-0) is similar to that known of Columbia (Col-0) ecotype. Restriction analysis and specific Southern blots of plastid DNA of L er-0, Cape Verdi Islands (Cvi-0), Col-0, Leiden (Le-0) and Stockholm (St-0) ecotypes revealed that, with respect to other ecotypes, only Cvi-0 lacks a BamHI site and gains a new HindIII site in the small single copy (SSC) region of plastid DNA. As in most plants, Northern blots for ndh transcripts revealed that SSC is expressed in Arabidopsis. Sequencing of a 1519b fragment around ndhG gene DNA confirmed that plastid DNA of Cvi-0 lacks a BamHI site (present in other ecotypes) at positions 46–51 down the initiation codon (ATG) of ndhG. BamHI digestions of the corresponding fragments amplified from plastid DNA of L er-0, Cvi-0 and L er-0 × Cvi-0 first 38 offsprings always showed maternal inheritance of plastid DNA.

Characterization of the genes, including that encoding the viral proteinase, contained in BamHI restriction fragment 9 of the pseudorabies virus genome.

We describe the nucleotide sequence, transcription pattern and open reading frames (ORFs) located on BamHI restriction fragment 9 (0.406-0.435 map units) in the unique long segment of the pseudorabies virus (PRV) genome. The fragment contains three nested genes with a common 3' end. The 5' ends of the corresponding 0.9, 1.7 and 3.3 kb mRNAs have been mapped. Fragment BamHI-9 contains three complete ORFs, ORF1, ORF2 and ORF2.5. ORF1, which is within the 3.3 kb transcript, encodes a protein with an apparent molecular mass of 60 kDa which is homologous to the product of the herpes simplex virus type 1 UL25 gene. The 1.7 kb mRNA contains ORF2, whose product is homologous to the herpesvirus proteinases, while the 0.9 kb transcript contains ORF2.5, which probably encodes the assembly protein precursor. ORF2 was identified as the PRV proteinase gene following expression in E. coli, using the product of ORF2.5 as the substrate protein.