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PubMed1997

Nested DNA inversion as a paradigm of programmed gene rearrangement.

Programmed gene rearrangements are employed by a variety of microorganisms, including viruses, prokaryotes, and simple eukaryotes, to control gene expression. In most instances in which organisms mediate host evasion by large families of homologous gene cassettes, the mechanism of variation is not thought to involve DNA inversion. Here we report that Campylobacter fetus, a pathogenic Gram-negative bacterium, reassorts a single promoter, controlling surface-layer protein expression, and one or more complete ORFs strictly by DNA inversion. Rearrangements were independent of the distance between sites of inversion. These rearrangements permit variation in protein expression from the large surface-layer protein gene family and suggest an expanding paradigm of programmed DNA rearrangements among microorganisms.

Bacterial Outer Membrane Proteins↗
PubMed2000

Rapid diagnosis of tuberculous pleural effusion using polymerase chain reaction.

Between October 1998 and September 1999, 98 patients with symptomatic exudative lymphocytic pleural effusion were enrolled in our study to evaluate the diagnostic sensitivity of polymerase chain reaction (PCR) assay. The mean age was 53.3 years ranging from 18 to 78 years. There were 61 men and 37 women. Pleural fluid was sent for gram staining, AFB staining, aerobic culture, culture of Mycobacterium tuberculosis on LJ media, and cytology. Additional fluid was used for a PCR-assay of the 16 S-23 S rRNA gene spacer sequences and for a nested PCR of the 16 S rRNA gene as a blind control. In cases of free-flow pleural tapping, histopathological analysis was done on three pleural biopsies. Overall etiologies comprised malignancy 53.1%, tuberculosis 36.7%, lymphoma 2.0% and chronic nonspecific inflammation 8.2%. The sensitivity and specificity of AFB-staining were 6% and 79%, respectively; while cultures on LJ media were 17% and 100%, respectively. The sensitivity of the PCR-assay was 50% (95% CI: 40 to 60%) and the specificity was 61% (95 CI: 52 to 71%). When PCR was nested, the sensitivity was 72% (95% CI: 63 to 81%) and specificity was 53% (95% CI: 43 to 63%). Two thirds (26 of 36) of tuberculous pleural effusion cases underwent pleural biopsy, and 62% were diagnosed by histopathology. There were no complications from thoracocentesis or pleural biopsy in any of the patients. We concluded that PCR assay was more sensitive than AFB staining and mycobacteria culture for diagnosis tuberculous pleural effusion but its specificity was quite low.

Adolescent↗
PubMed2000

The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumor suppressor genes. The International Lung Cancer Chromosome 3p21.3 Tumor Suppressor Gene Consortium.

We used overlapping and nested homozygous deletions, contig building, genomic sequencing, and physical and transcript mapping to further define a approximately 630-kb lung cancer homozygous deletion region harboring one or more tumor suppressor genes (TSGs) on chromosome 3p21.3. This location was identified through somatic genetic mapping in tumors, cancer cell lines, and premalignant lesions of the lung and breast, including the discovery of several homozygous deletions. The combination of molecular manual methods and computational predictions permitted us to detect, isolate, characterize, and annotate a set of 25 genes that likely constitute the complete set of protein-coding genes residing in this approximately 630-kb sequence. A subset of 19 of these genes was found within the deleted overlap region of approximately 370-kb. This region was further subdivided by a nesting 200-kb breast cancer homozygous deletion into two gene sets: 8 genes lying in the proximal approximately 120-kb segment and 11 genes lying in the distal approximately 250-kb segment. These 19 genes were analyzed extensively by computational methods and were tested by manual methods for loss of expression and mutations in lung cancers to identify candidate TSGs from within this group. Four genes showed loss-of-expression or reduced mRNA levels in non-small cell lung cancer (CACNA2D2/alpha2delta-2, SEMA3B [formerly SEMA(V), BLU, and HYAL1] or small cell lung cancer (SEMA3B, BLU, and HYAL1) cell lines. We found six of the genes to have two or more amino acid sequence-altering mutations including BLU, NPRL2/Gene21, FUS1, HYAL1, FUS2, and SEMA3B. However, none of the 19 genes tested for mutation showed a frequent (>10%) mutation rate in lung cancer samples. This led us to exclude several of the genes in the region as classical tumor suppressors for sporadic lung cancer. On the other hand, the putative lung cancer TSG in this location may either be inactivated by tumor-acquired promoter hypermethylation or belong to the novel class of haploinsufficient genes that predispose to cancer in a hemizygous (+/-) state but do not show a second mutation in the remaining wild-type allele in the tumor. We discuss the data in the context of novel and classic cancer gene models as applied to lung carcinogenesis. Further functional testing of the critical genes by gene transfer and gene disruption strategies should permit the identification of the putative lung cancer TSG(s), LUCA, Analysis of the approximately 630-kb sequence also provides an opportunity to probe and understand the genomic structure, evolution, and functional organization of this relatively gene-rich region.

Carcinoma, Non-Small-Cell Lung↗
PubMed1996

Rapid diagnosis of methicillin-resistant Staphylococcus aureus bacteremia by nested polymerase chain reaction.

OBJECTIVE: The purpose of this study was to establish a rapid and sensitive diagnostic method for methicillin-resistant Staphylococcus aureus (MRSA) bacteremia in postoperative patients. SUMMARY BACKGROUND DATA: As a result of diffusion and abuse of third-generation cephalosporin antibiotics in the 1980s in Japan, an outbreak of MRSA infection has been posed. In the field of surgery, severe postoperative infections with MRSA such as MRSA bacteremia, which may lead to multiple organ failure, have emerged with a high mortality. METHODS: Thirty-five patients with high fever (above 38.5 C) or watery diarrhea or both within 2 weeks after gastrointestinal major surgery and 6 healthy volunteers were examined. Nested polymerase chain reaction was used to detect mecA and toxic shock syndrome toxin-1 (TSST-1) genes in blood specimens. RESULTS: The mecA and TSST-1 genes were not detected in the blood samples of any of the six healthy volunteers. In all 12 samples from which MRSA colonies were isolated by blood culture, mecA and TSST-1 genes were detected. Although it took at least 48 hours to identify MRSA by the blood culture method, the presence of mecA and TSST-1 genes was determined by nested polymerase chain reaction method within only 3 to 4 hours after blood sampling. CONCLUSIONS: This method, as a sensitive and rapid monitoring system for MRS bacteremia, would be clinically beneficial for prevention of cross infection and for early determination of appropriate treatment for infected patients.

Bacteremia↗
PubMed2004

Identification of a novel box C/D snoRNA from mouse nucleolar cDNA library.

By construction and screen of mouse nucleolar cDNA library, a novel mammalian small nucleolar RNAs (snoRNA) was identified. The novel snoRNA, 70 nt in length, displays structural features typical of C/D box snoRNA family. The snoRNA possesses an 11-nt-long rRNA antisense element and is predicted to guide the 2'-O-methylation of mouse 28S rRNA at G4043, a site unknown so far to be modified in vertebrates. The comparison of functional element of snoRNA guides among eukaryotes reveals that the novel snoRNA is a mammalian counterpart of yeast snR38 despite highly divergent sequence between them. Mouse and human snR38 and other cognates in distant vertebrates were positively detected with slight length variability. As expected, the rRNA ribose-methylation site predicted by mouse snR38 was precisely mapped by specific-primer extension assay. Furthermore, our analyses show that mouse and human snR38 gene have multiple variants and are nested in the introns of different host genes with unknown function. Thus, snR38 is a phylogenetically conserved methylation guide but exhibits different genomic organization in eukaryotes.

Animals↗
PubMed1998

Genetic interactions and dosage effects of Polycomb group genes in mice.

In Drosophila and mouse, Polycomb group genes are involved in the maintenance of homeotic gene expression patterns throughout development. Here we report the skeletal phenotypes of compound mutants for two Polycomb group genes bmi1 and M33. We show that mice deficient for both bmi1 and M33 present stronger homeotic transformations of the axial skeleton as compared to each single Polycomb group mutant, indicating strong dosage interactions between those two genes. These skeletal transformations are accompanied with an enhanced shift of the anterior limit of expression of several Hox genes in the somitic mesoderm. Our results demonstrate that in mice the Polycomb group genes act in synergy to control the nested expression pattern of some Hox genes in somitic mesodermal tissues during development.

Animals↗
PubMed1995

Cloning and characterization of a novel NK cell-specific serine protease gene and its functional 5'-flanking sequences.

Rat natural killer cell Met-ase-1 (RNK-Met-1) is a 30,000 M(r) serine protease (granzyme) found in the cytolytic granules of CD3- large granular lymphocytes (LGL) with natural killer (NK) activity. To characterize the genomic sequences responsible for the CD3- LGL-restricted expression of this gene, we screened a rat genomic library with RNK-Met-1 cDNA, and obtained bacteriophage clones that contained the RNK-Met-1 gene. The RNK-Met-1 gene comprises 5 exons and spans approximately 5.2 kilobases (kb), exhibiting a similar structural organization to a class of CTL-serine proteases with protease catalytic residues encoded near the borders of exons 2, 3, and 5. The 5'-flanking region of the RNK-Met-1 gene contains a number of putative promoter and enhancer regulatory elements and shares several regions of homology with the 5'-flanking region of the mouse perforin gene. We have prepared nested deletions from approximately 3.3 kb of the 5'-flanking region of the RNK-Met-1 gene, and inserted these upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. These 5'-flanking RNK-Met-1-CAT constructs were transiently transfected into rat LGL leukemia, T-lymphoma, and basophilic leukemia cell lines. The transcriptional activity of the RNK-Met-1 5'-flanking region was strong, restricted to the RNK-16 LGL leukemia and controlled by several positive cis-acting regions spread over at least 3.3 kb. The longest and most active 5'-flanking region (-3341 to -33) was also used to drive specific expression of beta-galactosidase in RNK-16. These data are consistent with the NK cell-specific expression of RNK-Met-1 and suggest the potential utility of this gene promoter in the development of transgene models of NK cell biology in vivo.

Amino Acid Sequence↗
PubMed2002

Synthesis and expression of viral antigens in Vero cells persistently infected with Junin virus.

Two Vero cell lines persistently infected with XJCl3 and Cl67 strains of Junin virus and named V3 and V7, respectively, have been characterized with respect to the presence and expression of the nucleoprotein (N) and the glycoprotein precursor (GPC) viral genes. After the acute phase of infection, where a marked CPE and high titers of virus were obtained, JV persistently infected cells became morphologically undistinguishable from Vero cells and virus production dropped to undetectable levels. V3 and V7 were resistant to the superinfection with antigenically related viruses. This fact could not be attributed to the presence of defective interfering particles or non-infectious virus in the supernatant. Expression of N was consistently detected in both cultures and accumulation of two degradation products of N was evident during the late passages. Although no G1 (main surface glycoprotein) expression was observed, a marked fusogenic capacity was detected in both cultures indicating at least, the synthesis of a GPC derived fusogenic glycoprotein. Cell lysates from V3 and V7 subjected to RT-PCR, using specific primers for N gene, or to a nested RT-PCR using specific primers for GPC (G1 region) confirmed the presence of both viral genes. No viral DNA sequences could be detected in JV persistently infected cells.

Animals↗
PubMed1998

Nest building is a sexually selected behaviour in the barn swallow.

Females may use male nest building to assess male parental quality, and nest size would then be a sexually selected trait. In the barn swallow, Hirundo rustica, females select their partner by his tail length, a character believed to signal good genes. Both sexes participate in nest building, although male participation is negatively related to his attractiveness as reflected by tail length. We tested the hypothesis that nest building is a sexually selected trait: females paired with males of high parental quality (as shown by the male during nest building) may obtain a mate providing large amounts of parental investment, while, as has been shown previously, females mated to attractive (long-tailed) males will acquire mates with good genetic quality. Therefore, since nest building in barn swallows occurs after mating, we predicted a postmating sexual selection process by which the female invests differentially in reproduction depending on the male's nest-building effort (reflecting his willingness to invest in reproduction). The volume of material in a nest was related to the male's contribution to nest building and, in agreement with our hypothesis, in a multiple regression analysis, male tail length and nest material volume were negatively related to laying date and positively to female investment in reproduction (total number of eggs laid during the breeding season). Moreover, females paired with long-tailed males (which contribute very little to nest building), but using the same amount of nest material as females paired with short-tailed males, reduced the thickness of the nest and hence increased its capacity. Therefore, in the barn swallow two different traits appear to be sexually selected: tail length of males owing to the good genes process and nest-building ability owing to the good parent process. (c) 1998 The Association for the Study of Animal Behaviour.

Journal Article↗
PubMed2005

Vertebrate head development: segmentation, novelties, and homology.

Vertebrate head development is a classical topic lately invigorated by methodological as well as conceptual advances. In contrast to the classical segmentalist views going back to idealistic morphology, the head is now seen not as simply an extension of the trunk, but as a structure patterned by different mechanisms and tissues. Whereas the trunk paraxial mesoderm imposes its segmental pattern on adjacent tissues such as the neural crest derivatives, in the head the neural crest cells carry pattern information needed for proper morphogenesis of mesodermal derivatives, such as the cranial muscles. Neural crest cells make connective tissue components which attach the muscle fiber to the skeletal elements. These crest cells take their origin from the same visceral arch as the muscle cells, even when the skeletal elements to which the muscle attaches are from another arch. The neural crest itself receives important patterning influences from the pharyngeal endoderm. The origin of jaws can be seen as an exaptation in which a heterotopic shift of the expression domains of regulatory genes was a necessary step that enabled this key innovation. The jaws are patterned by Dlx genes expressed in a nested pattern along the proximo-distal axis, analogous to the anterior-posterior specification governed by Hox genes. Knocking out Dlx 5 and 6 transforms the lower jaw homeotically into an upper jaw. New data indicate that both upper and lower jaw cartilages are derived from one, common anlage traditionally labelled the "mandibular" condensation, and that the "maxillary" condensation gives rise to other structures such as the trabecula. We propose that the main contribution from evolutionary developmental biology to solving homology questions lies in deepening our biological understanding of characters and character states.

Animals↗
PubMed2002

Presence of European bat lyssavirus RNas in apparently healthy Rousettus aegyptiacus bats.

Apparently healthy Rousettus aegyptiacus bats were randomly chosen from a Dutch colony naturally infected with European bat lyssavirus subgenotype 1a (EBL1a). These bats were euthanised three months after the first evidence of an EBL1a infection in the colony. EBL1a genomic and antigenomic RNAs of the nucleoprotein gene were detected by nested reverse transcriptase PCR in 75% of the examined Rousettus aegyptiacus bats. The EBL1a RNAs of the nucleoprotein gene were detected mainly in brain tissues, but also in other organs. EBL1a messenger RNAs of the nucleoprotein gene and the glycoprotein gene were detected in brain tissues. The standard fluorescent antibody test revealed the presence of lyssavirus antigens in brain tissues from 7 (17.5%) Rousettus aegyptiacus bats. Furthermore, EBL1a could not be detected by virus isolation on murine neuroblastoma cells or by intracerebral inoculation of suckling mice. Neutralising antibodies directed against EBL1 were detected in 11% of the examined bats. This study shows that at least 85% of the apparently healthy Rousettus aegyptiacus bats must have been infected with EBL1a, and that these bats may survive from an EBL1a infection. Furthermore, the study supports the possibility of a long-term maintenance of EBL1a genome in Rousettus aegyptiacus bats.

Animals↗
PubMed1999

The orphan human pregnane X receptor mediates the transcriptional activation of CYP3A4 by rifampicin through a distal enhancer module.

Cytochrome P-450 3A4 (CYP3A4), the predominant cytochrome P-450 expressed in adult human liver, is subject to transcriptional induction by a variety of structurally unrelated xenobiotics, including the antibiotic rifampicin. The molecular mechanisms underlying this phenomenon are poorly understood. We transfected a human liver-derived cell line (HepG2) with various CYP3A4-luciferase reporter gene constructs containing a nested set of 5'-deletions of the CYP3A4 5'-flanking region. Rifampicin-inducible transcription of the reporter gene was observed only with the longest construct, which encompassed bases -13000 to +53 of CYP3A4 (3-fold induction). The responsive region was functional regardless of its position or orientation relative to the proximal promoter of CYP3A4 and was capable of conferring rifampicin-inducible expression on a heterologous promoter. Further deletion mutants localized the induction to bases -7836 to -7607. In vitro DNase I footprint analysis of this region revealed four protected sites (FP1, FP2, FP3, and FP4). Two of these sites, FP3 (bases -7738 to -7715) and FP4 (bases -7698 to -7682), overlapped binding motifs for the orphan human pregnane X receptor (hPXR). Cotransfection of responsive constructs with a hPXR expression vector substantially increased the rifampicin-inducibility to approximately 50-fold. In addition, the rifampicin-responsive constructs were strongly activated by a range of CYP3A inducers. Finally, we demonstrate cooperativity between elements within the distal enhancer region and cis-acting elements in the proximal promoter of CYP3A4. Our results provide evidence for the existence of a potent enhancer module, 8 kb distal to the transcription start point, which mediates the transcriptional induction of CYP3A4 by activators of hPXR.

3T3 Cells↗
PubMed2005

Improved detection of bovine coronavirus N gene in faeces of calves infected naturally by a semi-nested PCR assay and an internal control.

Bovine coronavirus (BCoV), a positive sense single-stranded RNA virus, is an important causative agent of neonatal diarrhoea in calves from beef and dairy cattle worldwide. The routine detection and diagnosis of BCoV have been mainly dependent on assays with low sensitivity. The aim of the present study was to develop and evaluate a semi-nested PCR (SN-PCR) to amplify a 251bp fragment of BCoV N gene from fresh (n=25) and frozen (n=25) diarrhoeic faecal samples of naturally infected calves. To improve detection of BCoV in faecal samples by the SN-PCR an internal control was developed, and the results were compared with a conventional RT-PCR assay. The rates of positive samples by SN-PCR and RT-PCR were 24% (12/50) and 8% (4/50), respectively (K=0.43). Only fresh samples were positive in RT-PCR while the SN-PCR detected BCoV in both fresh and frozen faecal samples. The sensitivity of SN-PCR was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) that was detected until 10(-7) dilution. The specificity of the amplicons was assessed by restriction fragment length polymorphism and sequence analysis. The inclusion of an internal control provides a way to detect assay inhibition in faecal samples and failure of nucleic acid extraction that allow reduction of the number of false-negative results.

Animals↗
PubMed2003

Nested PCR for detection of mutans streptococci in dental plaque.

AIMS: Mutans streptococci such as Streptococcus mutans and Streptococcus sobrinus have been implicated in human dental caries. In an attempt to develop a rapid and sensitive method for detecting Strep. mutans and Strep. sobrinus in dental plaque, a nested PCR amplification based on the 16S rRNA gene was employed. METHODS AND RESULTS: A universal set of PCR primers for bacterial 16S rRNA gene was introduced for the first PCR, and then two sets of primers specific for the 16S rRNA gene sequences of either Strep. mutans or Strep. sobrinus were used for the second PCR. Eighteen plaque samples were analyzed, and a nested PCR was shown to be more sensitive for detecting Strep. mutans and Strep. sobrinus than direct PCR. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The 16S rRNA gene-based nested PCR method is a rapid and sensitive method for the detection of mutans streptococci, and may also be suitable for carrying out large-scale studies on the cariogenicity of mutans streptococci.

Adult↗
PubMed1994

Identification of a natural cycle involving Rickettsia typhi infection of Monopsyllus sciurorum sciurorum fleas from the nests of the fat dormouse (Glis glis).

In a mountainous area in the Dinaric Beech-Fir Forest of southern Slovenia, summer nests of the European fat dormouse (Glis glis) were collected. From these dormouse nests, 180 Monopsyllus sciurorum sciurorum fleas were examined by polymerase chain reaction with primers for the Rickettsia citrate synthase gene. Samples from one nest yielded the expected 381 base pair DNA product. The origin of the DNA product was identified as Rickettsia typhi by AluI restriction fragment length polymorphism analysis. Inoculation of the triturated positive fleas into Vero cell culture resulted in the cultivation of a rickettsia which reacted with polyclonal and species-specific monoclonal antibodies for R. typhi. The widespread distribution of this sylvatic flea species in nearly all of Europe as well as in the Middle East and its presence on other mammalian and avian hosts suggests that R. typhi might exist in unrecognised enzootic cycles. Further investigations are needed to determine the extent of these cycles in Europe and the potential occurrence of human infections.

Animals↗
PubMedDate not supplied

Antigenic and genomic relatedness of turkey-origin coronaviruses, bovine coronaviruses, and infectious bronchitis virus of chickens.

In earlier studies in our laboratory, we found that bovine coronavirus (BCV) was pathogenic for 1-day-old turkey poults. This finding prompted us to study the antigenic and genomic relatedness of turkey origin coronaviruses (TOCVs) to BCV. A one-step reverse transcription (RT)-polymerase chain reaction (PCR) targeting a 730-base pair fragment of the nucleocapsid (N) gene of BCV and a nested PCR targeting a 407-base pair fragment of the N gene were used in an attempt to detect TOCV from North Carolina, Indiana, and a prototype turkey coronavirus (TCV) obtained from the American Type Culture Collection. Both the one-step RT-PCR and the nested PCR amplified cell culture-passaged isolates of calf diarrhea strains of BCV but none of the 15 tested TOCVs or transmissible gastroenteritis coronavirus of swine. TOCVs also did not cross-react in a BCV antigen-capture (AC) enzyme-linked immunosorbent assay (ELISA) system with monoclonal antibodies (MAbs) against N, spike glycoprotein, and hemagglutinin esterase glycoprotein proteins of BCV as coating antibodies. The same TOCVs could be detected with primers designed from the genome of infectious bronchitis virus (IBV) of chickens. These primers amplified a 1082-base pair region spanning portions of the membrane glycoprotein (M) and N protein genes of IBV and TCV. The TOCVs also cross-reacted in an AC-ELISA with MAbs against the M and subunit 2 of spike glycoprotein of IBV.

Animals↗
PubMed2003

Hox genes as synchronized temporal regulators: implications for morphological innovation.

In vertebrates, clusters of Hox genes express in a nested and hierarchical fashion to endow the embryo's segments with discrete identities. Later in development, members of the same gene family are employed again to pattern the limb, intestinal, and reproductive systems. A careful analysis of the morphologies of Hox mutant mice suggests that the genes provide qualitatively different cues during the specification of segments than they do during the development of more recently derived structures. In addition to the regulatory differences noted by others, the activity of Hox genes during specification of the vertebrate metameres in some recent deletion experiments is inconsistent with a role for them as strictly spatial determinants. On the contrary, the phenotypes observed are suggestive of a role for them as elements of a generic time-keeping mechanism. By contrast, the specification of more recent evolutionary structures appears to be more spatial and gene-specific. These differences in role and effect may suggest some simple mechanisms by which the Hox clusters operate, and rules by which gene networks can be diverted to create new structures over the course of evolution. Specific predictions and experiments are proposed.

Animals↗