Polymerase chain reaction to detect infectious laryngotracheitis virus in conjunctival swabs from experimentally infected chickens

The polymerase chain reaction (PCR) was standardized and assessed as a potential diagnostic test for infectious laryngotracheitis using conjunctival swabs collected from experimentally infected chickens. Polymerase chain reaction primers based on the sequence of a 1.1-kb BamHI restriction enzyme fragment of the Ontario 1598 (Ont 1598) strain of infectious laryngotracheitis virus were selected and 300 fg of purified viral DNA were detected by ethidium bromide staining of agarose gels or 30 fg were detected by DNA hybridization. At least five different strains (Ont 1598, ATCC N-71851, LT-IVAX, Laryngo-Vac, and a local strain 322) were amplified whereas other avian pathogens and uninfected cell cultures tested negative. Swabs collected from experimentally infected chickens were analyzed by both PCR and virus isolation on various days postinfection. A comparison of virus isolation to PCR indicated that, in the mid-postinfection phase, PCR and virus isolation appeared to be comparable with a kappa value of greater than 0.8. The polymerase chain reaction was shown to be the better test later in infection, when clinical recovery had occurred.     

The elite athlete and exercise in pregnancy

To construct an effective recombinant Marek's disease virus type 1 (MDV1), we localized a stable insertion site for expression of the Escherichia coli lacZ gene near or within the short inverted repeats of MDV1 strain K554 DNA. A stable recombinant MDV1 was obtained by deleting the junction region between the short unique sequence (Us) and the internal short inverted repeat (IRs). The recombinant MDV1 replicated in cultured cells as well as the parental viral DNA. Antibodies against both MDV1 antigen and beta-galactosidase encoded by the lacZ gene were detected in the sera of chickens immunized with the virus, and persisted for at least 16 weeks. Moreover, the recombinant virus conferred protection upon chickens against a challenge with virulent MDV1. These results demonstrated that the Us-IRs junction region is an effective site for the insertion of foreign genes from which to construct a polyvalent live vaccine for poultry. Analysis of the Us-IRs junction region which was deleted from the parental MDV1 indicated that there is a tandem direct repeat of a 220-bp exists within the short internal and terminal inverted repeats of avirulent MDV1 K554 strain DNA. The 220-bp sequence was well conserved among DNAs from various strains. The number of the repeat units may differ between the IRs and TRs or among various MDV1 strain DNAs.     

Physical mapping of the linear plasmid pSLA2-L and localization of the eryAI and actI homologs

The 200-kb linear plasmid pSLA2-L was suggested to be involved in the production of lankamycin and lankacidin in Streptomyces rochei 7434AN4. In this study, we have constructed a physical map for 23 PstI fragments of pSLA2-L, the sum of which was 206 kb. Detailed restriction maps for both ends of pSLA2-L revealed the presence of terminal inverted repeats, the size of which was found to be 2.1 kb by cloning and sequencing of the end-points. Hybridization experiments using two polyketide biosynthetic genes, eryAI and actI, located their homologous regions on PstI fragments A and I, respectively.     

Physical and genetic chromosomal map of an M type 1 strain of Streptococcus pyogenes

A physical map of the chromosome of an M type 1 strain of Streptococcus pyogenes was constructed following digestion with three different restriction enzymes, SmaI, SfiI, and SgrAI, and separation and analysis of fragments by pulsed-field gel electrophoresis. The genome size of this strain was estimated to be 1,920 kb. By employing Southern hybridization and PCR analysis, 36 genes were located on the map.     

Screening for germ-line rearrangements and regulatory mutations in BRCA1 led to the identification of four new deletions

Pogo is a transposable element with short terminal inverted repeats. It contains two open reading frames that are joined by splicing and code for the putative pogo transposase, the sequence of which indicates that it is related to the transposases of members of the Tc1/mariner family as well as proteins that have no known transposase activity including the centromere binding protein CENP-B. We have shown that the N-terminal region of pogo transposase binds in a sequence-specific manner to the ends of pogo and have identified residues essential for this. The results are consistent with a prediction that DNA binding is due to a helix-turn-helix motif within this region. The transposase recognises a 12 bp sequence, two copies of which are present at each end of pogo DNA. The outer two copies occur as inverted repeats 14 nucleotides from each end of the element, and contain a single base mismatch and indicate the inverted repeats of pogo are 26 nucleotides long. The inner copies occur as direct repeats, also with a single mismatch.     

The physical map of the chloroplast DNA from Korean ginseng (Panax ginseng C.A. Meyer)

To compare the gene order of the chloroplast genome among dicotyledonous plants, we constructed a physical map of chloroplast DNA (cpDNA) of Korean ginseng (Panax ginseng C.A. Meyer) with four restriction enzymes, BamHI, HindIII, EcoRI, and PstI. The restriction enzyme recognition sites of the physical map were also confirmed by Southern hybridization of total ginseng cpDNA with homologous and heterologous probes. The cpDNA of Korean ginseng was determined as a circular molecule with a total size of about 154 kb, which contain two inverted repeats of 23 kb each that disrupt the rest of the molecule into a large (90 kb) and a small single copy region (18 kb). The genome structure of Korean ginseng cpDNA was similar in size and gene order to that of tobacco cpDNA. The cpDNA of Korean and American ginseng (P. quinquefolius) showed very similar restriction patterns.     

A region of Haemophilus influenzae Rd hybridizes with repetitive human DNA

A 17.7 kbp EcoR1 fragment of Haemophilus influenzae Rd (wild-type strain) DNA containing repeats has been cloned together with the DNA repair gene uvr3. The region containing repetitive DNA was subcloned on a 13 kbp EcoR1 fragment (pHi13). The insert hybridizes with restriction enzyme digests of H. influenzae Rd and H. parainfluenzae and exhibits a repetitive pattern. Discrete bands superimposed upon a polydisperse background were observed when pHi13 hybridizes with restriction digests of human DNA. The hybridization patterns indicate that H. parainfluenzae and human DNA contain cross-hybridizing sequences. The repeats of pHi13 are present on a 2.6 kbp PvuII fragment.     

A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP

Partial digestion of DNA fragments is a standard procedure for subcloning analysis and for generating restriction maps. We have developed a novel method to generate a partial digestion for any DNA fragment that can be amplified by PCR. The method involves the incorporation of 5-methyl-dCTP into the PCR product to protect most of the restriction sites. As a result, complete digestion of the modified PCR products with a 5-methyl-dCTP-sensitive enzyme will produce an array of restriction fragments equivalent to a partial restriction enzyme digestion reaction done on unmethylated PCR products. This method reduces the time and material needed to produce partially-digested DNA fragments by traditional methods. Furthermore, using fluorescein-labeled primers in the reaction, we were able to detect the fluorescein-labeled end fragments resulting from the enzyme digestion using a fluorimager or anti-fluorescein-AP antibody and thus determine the restriction maps.     

Transcriptional control of the equine herpesvirus 1 immediate early gene

Transient expression assays measuring induction of an equine herpesvirus 1 (EHV-1) immediate early (IE) promoter construct, pIE beta gal, were used to examine trans-induction of the IE gene (IR1) promoter by superinfection with intact EHV-1 (Kentucky A strain), uv-inactivated EHV-1, or EHV-1 stocks highly enriched for defective interfering particles (DIPs), and by cotransfection with plasmids containing EHV-1 DNA fragments. Our results confirm reports by others in that the IE promoter can be induced by both intact and uv-inactivated EHV-1 and provide evidence that DIP-rich virus stocks also possess transinducing activity. Furthermore, IE promoter activity was induced by cotransfection of the promoter construct with either of two plasmids containing restriction enzyme DNA fragments that span ORF12, the putative EHV-1 homolog of the herpes simplex virus 1 (HSV-1) alpha-trans-inducing factor (alpha-TIF) gene, UL48, or by cotransfection with an isolated DNA fragment containing only ORF12, while no transactivation was associated with plasmids linearized by restriction enzyme digestion at a single site within ORF12. These data indicate that, despite its predicted lack of an acidic carboxy terminus (a region essential in HSV-1 alpha-TIF for trans-inducing activity), the EHV-1 ORF12 product is a functional alpha-TIF.     

A contingency-based approach for assessing policies on aging

The analysis of genomic DNA fragment patterns has revealed as a powerful tool for strain discrimination in Staphylococcus aureus; for use as an epidemiological marker, stability during the course of an outbreak is an essential prerequisite. Genomic DNA fragment patterns (SmaI restriction, pulsed-field electrophoresis) of four different epidemic MRSA strains were compared along with intra- and interhospital and country-wide spread over more than 12 months in Germany. Strain I was isolated from infections in 8 hospitals. In one hospital a subclone arised which differed from the original strain by 4 fragments. Strain II was spread among 4 hospitals, isolates from three of these hospitals exhibited a variability of one to three fragments in the 150-200 kb range. Two hospitals in the Hannover-area were affected by strain III; in 17 isolates of this strain a variability up to three fragments was found in the 170-200 kb range. Strain IV was isolated from 19 cases of infections in 3 hospitals in Berlin. The fragment patterns were completely stable. When S. aureus strains are typed by genomic DNA fragment patterns, a variability in a definite range of molecular masses during the course of an epidemic should be taken into consideration.     

Lead intoxication in children with pervasive developmental disorders

Ten clinical isolated strains of Human cytomegalovirus (HCMV) were obtained from 73 urine specimens of different people. Isolations from urine were carried out in human embryolung fibroblasts. Viral isolates were passaged four times. HCMV DNAs of laboratory strain AD169 and 10 clinical isolated strains were extracted with Hirt method, digested with each of the restriction enzymes EcoRI, Hind II. Comparison of restriction fragment length polymorphism (RFLP) of AD169 and isolated strains were made by hybridizing digested DNA with 32P labeled with HCMV Hind II cloned subgenomic fragments as the probe (pCM1035, pCM1015). pCM1035 is located in the joining region between the long(L) and short (S) unique sequences of the virus (L-S junction) pCM1015 is located in the terminal sequences of the virus. The results showed the genomic high degree of homology existed among all strains and the variable restriction site was in the L-S junction and terminal portion. The RFLP patterns of the clinical isolates which did not have relation in epidemiology were different, but the patterns of clinical isolates related in epidemiology were quite similar. Polymorphism frequently occurred in this case of EcoRI digested fragment hybridized with the probe of pCM1035. Southern hybridization of HCMV isolations is useful to researches into the molecular epidemiology and pathogenesis of HCMV infection.     

Characterization of IS1547, a new member of the IS900 family in the Mycobacterium tuberculosis complex, and its association with IS6110

Unlike classically defined insertion sequence (IS) elements, which are delimited by their inverted terminal repeats, some IS elements do not have inverted terminal repeats. Among this group of atypical IS elements, IS116, IS900, IS901, and IS1110 have been proposed as members of the IS900 family of elements, not only because they do not have inverted terminal repeats but also because they share other features such as homologous transposases and particular insertion sites. In this study, we report a newly identified IS sequence, IS1547, which was first identified in a clinical isolate of Mycobacterium tuberculosis. Its structure, insertion site, and putative transposase all conform with the conventions of the IS900 family, suggesting that it is a new member of this family. IS1547 was detected only in isolates of the M. tuberculosis complex, where it had highly polymorphic restriction fragment length polymorphism patterns, suggesting that it may be a useful genetic marker for identifying isolates of the M. tuberculosis complex and for distinguishing different strains of M. tuberculosis. ipl is a preferential locus for IS6110 insertion where there are eight known different insertion sites for IS6110. Surprisingly, the DNA sequence of ipl is now known to be a part of IS1547, meaning that IS1547 is a preferential site for IS6110 insertion.     

Postnatal development of respiratory function in lambs studied serially between birth and 8 weeks

The molecular structure of the telomeric region at the left arm of the second chromosome of the mosquito Anopheles gambiae has been determined in the transformed strain G418 that contains a pUChsneo transgene attached at the 2L chromosome end, and in the Pink eye laboratory strain (PE). Both strains contain the same complex satellite positioned distal to a unique region. FIGE mapping of the telomeric region of the PE strain revealed distinct DNA fragment lengths that segregated with individual chromosomes. Genomic DNA fragments were cloned from the 2L telomeric region, which accounted for about half of 2L chromosomes in the PE population. In all three cases studied, long fragments of different middle repetitive sequences were found attached to the distal ends of the 2L satellite. We propose that random fragments of DNA may be occasionally added during recombination between complex satellite repeats at the chromosome ends.     

Management of mania in the elderly: an update

Twenty-eight genetic loci have been physically mapped to specific large restriction fragments of the Streptococcus mutans GS-5 chromosome by hybridization with probes of cloned genes or, for transposon-generated amino acid auxotrophs, with probes for Tn916. In addition, restriction fragments generated by one low-frequency-cleavage enzyme were used as probes to identify overlapping fragments generated by other restriction enzymes. The approach allowed construction of a low resolution physical map of the S. mutans GS-5 genome using restriction enzymes ApaI (5'-GGGCC/C), SmaI (5'-CCC/GGG), and NotI (5'-GC/GGCCGC).     

The a sequence is dispensable for isomerization of the herpes simplex virus type 1 genome

The herpes simplex virus type 1 (HSV-1) genome consists of two components, L (long) and S (short), that invert relative to each other during productive infection to generate four equimolar isomeric forms of viral DNA. Recent studies have indicated that this genome isomerization is the result of DNA replication-mediated homologous recombination between the large inverted repeat sequences that exist in the genome, rather than site-specific recombination through the terminal repeat a sequences present at the L-S junctions. However, there has never been an unequivocal demonstration of the dispensability of the latter element for this process using a recombinant virus whose genome lacks a sequences at its L-S junctions. This is because the genetic manipulations required to generate such a viral mutant are not possible using simple marker transfer, since the cleavage and encapsidation signals of the a sequence represent essential cis-acting elements which cannot be deleted outright from the viral DNA. To circumvent this problem, a simple two-step strategy was devised by which essential cis-acting sites like the a sequence can be readily deleted from their natural loci in large viral DNA genomes. This method involved initial duplication of the element at a neutral site in the viral DNA and subsequent deletion of the element from its native site. By using this approach, the a sequence at the L-S junction was rendered dispensable for virus replication through the insertion of a second copy into the thymidine kinase (TK) gene of the viral DNA; the original copies at the L-S junctions were then successfully deleted from this virus by conventional marker transfer. The final recombinant virus, HSV-1::L-S(delta)a, was found to be capable of undergoing normal levels of genome isomerization on the basis of the presence of equimolar concentrations of restriction fragments unique to each of the four isomeric forms of the viral DNA. Interestingly, only two of these genomic isomers could be packaged into virions. This restriction was the result of inversion of the L component during isomerization, which prevented two of the four isomers from having the cleavage and encapsidation signals of the a sequence in the TK gene in a packageable orientation. This phenomenon was exploited as a means of directly measuring the kinetics of HSV-1::L-S(delta)a genome isomerization. Following infection with virions containing just the two packaged genomic isomers, all four isomers were readily detected at a stage in infection coincident with the onset of DNA replication, indicating that the loss of the a sequence at the L-S junction had no adverse effect on the frequency of isomerization events in this virus. These results therefore validate the homologous recombination model of HSV-1 genome isomerization by directly demonstrating that the a sequence at the L-S junction is dispensable for this process. The strategy used to remove the a sequence from the HSV-1 genome in this work should be broadly applicable to studies of essential cis-acting elements in other large viral DNA molecules.     

Differential effects of X-irradiation and cyclosporin-A administration on the thymus with respect to the generation of cyclosporin-A-induced autoimmunity

A new insertion sequence in Streptococcus pneumoniae was identified as a 1435-bp segment of the genome containing 24-bp terminal inverted repeats and flanked by 8-bp direct repeats. A copy of the element, named IS1167, adjacent to the comAB genes was sequenced; seven additional copies were found in the genome of strain CP1200 and relatives descended from strain R36A. Among 22 independent pneumococcal isolates, 11 contained copies of elements hybridizing to IS1167 in nine distinct restriction fragment patterns, with 3-12 copies each. The bulk of the element was occupied by two overlapping open reading frames, encoding basic proteins which together exhibited strong similarity to the full length of the putative transposase of the staphylococcal transposable element, IS1181, as well as significant similarity to those of seven additional known or putative insertion sequences related to the mycobacterial element, IS1096.     

9-(4-Hydroxybutyl)-N2-phenylguanine (HBPG), a thymidine kinase inhibitor, suppresses herpes virus reactivation in mice

In cells of the nervous system, which have little or no cellular thymidine kinase, the pharmacologic inhibition of viral thymidine kinase may prevent the reactivation of herpes virus, which requires phosphorylated thymidine for replication. We tested a newly synthesized inhibitor of viral thymidine kinase, 9-(4-hydroxybutyl)-N2-phenylguanine (HBPG) for its capacity to suppress the reactivation of herpes simplex virus type 1 (HSV-1) in vivo. Mice, latently infected with McKrae strain HSV-1, were treated with intraperitoneal injections of HBPG in a corn oil vehicle (200 mg/kg every 3 h for a total of ten doses), and subjected to hyperthermic stress to stimulate viral reactivation immediately before the third treatment. Three h after the last treatment, the mice were sacrificed, and the presence of infectious virus was determined by culture of ocular surface swabs and trigeminal ganglionic homogenates. Additionally, viral DNA in ganglionic extracts was analyzed by quantitative PCR. Controls included latently infected, stressed animals receiving injections of corn oil vehicle only, and latently infected, drug- and vehicle-treated, unstressed animals. HBPG had a statistically significant inhibitory effect on hyperthermia-induced viral reactivation. Homogenates of trigeminal ganglia and ocular surface swabs from HBPG-treated animals were less likely to contain infectious virus than those of infected, vehicle-treated, stressed controls (P < 0.005, ANOVA). Unstressed controls showed no reactivation. Quantitation of viral DNA in ganglionic extracts demonstrated a 100-fold reduction in the amount of viral DNA in the ganglia of HBPG-treated animals, compared with vehicle-treated controls (P < 0.05, ANOVA). The results indicate that HBPG has an inhibitory effect when given systemically for the suppression of herpes virus reactivation in mice.     

Synthesis of a novel sialic acid derivative (sialylphospholipid) as an antirotaviral agent

A novel sialylphospholipid (SPL) was synthesized from N-acetylneuraminic acid (NeuAc) and phosphatidylcholine (PC) by a chemical and enzymatic method and evaluated as an inhibitor of rotavirus. PC and 1,8-octanediol were conjugated by transesterification reaction of Streptomyces phospholipase D (PLD) under a water-chloroform biphasic system to afford phosphatidyloctanol, which was condensed with a protected 2-chloro-2-deoxyneuraminic acid derivative by using silver trifluoromethanesulfonate as an activator in chloroform and converted, after deprotection, to SPL. Rhesus monkey kidney cells (MA-104) were incubated with simian (SA-11 strain) and human (MO strain) rotaviruses in the presence of SPL, and the cells infected were detected indirectly with anti-rotavirus antibody. SPL showed dose dependent inhibition against both virus strains. The concentrations required for 50% inhibition (IC50) against SA-11 and MO were 4.35 and 16.1 microM, respectively, corresponding to 10(3)- and 10(4)-fold increases in inhibition as compared to monomeric NeuAc.     

The product of the herpes simplex virus type 1 UL25 gene is required for encapsidation but not for cleavage of replicated viral DNA

The herpes simplex virus type 1 (HSV-1) UL25 gene contains a 580-amino-acid open reading frame that codes for an essential protein. Previous studies have shown that the UL25 gene product is a virion component (M. A. Ali et al., Virology 216:278-283, 1996) involved in virus penetration and capsid assembly (C. Addison et al., Virology 138:246-259, 1984). In this study, we describe the isolation of a UL25 mutant (KUL25NS) that was constructed by insertion of an in-frame stop codon in the UL25 open reading frame and propagated on a complementing cell line. Although the mutant was capable of synthesis of viral DNA, it did not form plaques or produce infectious virus in noncomplementing cells. Antibodies specific for the UL25 protein were used to demonstrate that KUL25NS-infected Vero cells did not express the UL25 protein. Western immunoblotting showed that the UL25 protein was associated with purified, wild-type HSV A, B, and C capsids. Transmission electron microscopy indicated that the nucleus of Vero cells infected with KUL25NS contained large numbers of both A and B capsids but no C capsids. Analysis of infected cells by sucrose gradient sedimentation analysis confirmed that the ratio of A to B capsids was elevated in KUL25NS-infected Vero cells. Following restriction enzyme digestion, specific terminal fragments were observed in DNA isolated from KUL25NS-infected Vero cells, indicating that the UL25 gene was not required for cleavage of replicated viral DNA. The latter result was confirmed by pulsed-field gel electrophoresis (PFGE), which showed the presence of genome-size viral DNA in KUL25NS-infected Vero cells. DNase I treatment prior to PFGE demonstrated that monomeric HSV DNA was not packaged in the absence of the UL25 protein. Our results indicate that the product of the UL25 gene is required for packaging but not cleavage of replicated viral DNA.