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Biomedical subjects

M G Romanelli

Publications and source records attributed to M G Romanelli.

At least 19 recordsLinked to original sources
PubMed2001

Cloning and functional characterization of the human heterogeneous nuclear ribonucleoprotein type I promoter.

We have cloned and functionally characterized a portion of the human hnRNP I (heterogeneous nuclear ribonucleoprotein type I) gene containing the promoter elements. HnRNP I is an alternative splicing modulator of tissue-specific transcripts that is expressed in three different isoforms. The DNA sequence at the transcription start site, identified by 5'-rapid amplification of cDNA ends, shows a high 'GC' content, lacks canonical TATA sequences and contains multiple putative Sp1 and NF1 transcription factor-binding sites, a GATA box and a CAAT box. By means of a chloramphenicol acetyltransferase reporter construct and deletion analyses, we have identified two regions between -770 bp and -206 bp that had a positive effect on expression activity in HeLa cells.

Base Sequence↗
PubMed2000

Organization of the human gene encoding heterogeneous nuclear ribonucleoprotein type I (hnRNP I) and characterization of hnRNP I related pseudogene.

The human gene hnRNPI encoding the heterogeneous nuclear ribonucleoprotein type I, an alternative splicing modulator of tissue-specific transcripts, also known as PTB (polypyrimidine tract-binding protein), was recently mapped on chromosome 14, as well as on chromosome 19, suggesting that two closely related copies of the same gene might exist in the human genome. We report here that the gene localized on chromosome 14 corresponds to a highly homologous processed pseudogene related to hnRNPI gene (psihnRNPI). Analysis by RT-PCR and by EST database comparison indicates that psihnRNPI is not expressed. In this report we have also analyzed the organization of the actual hnRNPI gene localized on chromosome 19. The DNA sequence at the intron-exon boundaries unveiled the possible mechanism by which three isoforms of the protein (namely hnRNPI, PTB2 and PTB3) are generated by means of alternative splicing of the same hnRNPI gene transcript.

Amino Acid Sequence↗
PubMed2000

The response of pigs inoculated with a thymidine kinase-negative (TK-) pseudorabies virus to challenge infection with virulent virus.

12 Large-White-Landrace piglets were subdivided in four groups of 3 and housed in separate units. The piglets of three groups were inoculated with the 86/27V 6C2 thymidine kinase negative (TK-) mutant of pseudorabies virus (PRV), by different routes. A second inoculation with the same mutant was given to the pigs 21 days later. The animals of a fourth group were left as uninoculated controls. 21 days following the second inoculation with the TK- mutant all pigs were challenge infected with the virulent PRV. On post challenge day (PCD) 30 all pigs were killed and samples for virus detection and histology were taken from several organs. The inoculated TK- mutant of PRV did not induce any ill effects in the pigs except a transient febrile reaction in some animals. Virus was recovered from nasal swabbings from one pig 2 days after the first inoculation of the mutant. After challenge exposure with virulent PRV, the TK- mutant-inoculated pigs were apparently protected, whereas the control pigs all were severely affected and recovered very slowly over 3 weeks. Virus was isolated from the nasal swabbings from the TK- mutant-inoculated pigs on PCDs 2 and 4, whereas the nasal swabbings from the control piglets were all positive for virus from PCD 2 through PCD 10. DNA analysis of the virus recovered showed a pattern identical to that of the virulent PRV. Histologic lesions were found in the respiratory and the central nervous systems, however, the lesions in the TK- mutant-inoculated pigs were much milder compared to those registered for the control pigs. Virus was not isolated from any of the tissue samples that were tested, but viral DNA with sequences typical of PRV genome was detected by PCR in all samples of trigeminal ganglia from either the TK- mutant-inoculated pigs or from the controls.

Administration, Intranasal↗
PubMed2000

A study of the ability of a TK-negative and gI/gE-negative pseudorabies virus (PRV) mutant inoculated by different routes to protect pigs against PRV infection.

The capacity of a TK-negative (TK-) and gI/gE-negative (gI/gE-) pseudorabies virus (PRV) mutant to protect pigs against Aujeszky's disease carried out by experimental infection with a virulent PRV strain, was tested. There were three groups, each of four susceptible pigs which were inoculated twice by two different schedules. Group 1 received the modified virus by the intradermal (first inoculation)-intramuscular (second inoculation) routes; group 2 was treated by the intranasal (first inoculation)-intramuscular (second inoculation) routes. The third group was left untreated as the control. All of the pigs were challenged intranasally with a virulent PRV strain and they were subsequently injected with dexamethasone. Two pigs in each group were necropsied on days 5 and 15 after dexamethasone inoculation. The challenge exposure resulted in mild clinical signs, increase in growth and a shorter period of virus shedding in vaccinated pigs, whereas the control group showed severe signs of Aujeszky's disease. No difference in the titre of the virulent virus which was excreted by pigs of all three groups, was observed and all animals seroconverted. Both the mutant strain and the wild-type virus established a latent infection although only the latter was reactivated and shed. Slight lesions were observed in target tissues of the vaccinated animals and no significant differences were detected between the two inoculation schedules.

Administration, Intranasal↗
PubMed1998

Experimental infection of pigs with a thymidine kinase negative strain of pseudorabies virus.

Sixteen 20 day old pigs, devoid of neutralizing antibody to pseudorabies virus (PRV), were divided into two groups of eight, an the animals of each group were housed in a separate unit. In each group 6 pigs were inoculated intranasally with the thymidine kinase (TK-) mutant (Group 1) or the field strain of PRV (Group 2), each pig receiving an inoculum of 4 ml. The remaining 2 pigs in each group served as uninoculated controls. The only clinical sign observed in the pigs of Group 1 was a transient febrile reaction, in the case of six pigs inoculated with the TK- mutant of PRV, whereas no signs of disease were seen in the uninoculated controls. The virus was isolated from the 6 infected pigs of the group only on post infection day (PID) 2, whereas it was never isolated from the controls. By contrast, the pigs of Group 2, had a severe clinical response and one, among those that were inoculated with the field strain of the PRV, died on PID 9. Virus was consistently isolated from all pigs of Group 2, inoculated and control. On PID 30 all pigs, i.e. the 8 of Group 1 and 7 of the Group 2 which survived to the infection, were subjected to dexamethasone (DMS) treatment. After DMS treatment virus was never isolated from the nasal swabbings obtained from the pigs of Group 1, whereas it was consistently isolated from pigs of Group 2. After 30 d from the start of DMS treatment the pigs were killed and several tissues were collected from each pig for virus detection, by isolation in tissue culture and by PCR analysis. At necropsy no lesions were found in pigs of Group 1, whereas acute pneumonia and gliosis in the trigeminal ganglia were observed in pigs of Group 2. Virus was never isolated from any of the tissues taken from pigs of both, Group 1 and Group 2, nevertheless sequences of PRV were detected by PCR analysis in the trigeminal ganglia of the pigs of both Groups.

Animals↗
PubMed1997

Sequence determinants for hnRNP I protein nuclear localization.

hnRNP I, also referred to as polypyrimidine tract binding protein, is one of the proteins associated with nascent pre-mRNA in the heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. As for all karyophilic proteins, the nuclear import of hnRNP proteins requires specific sequence determinants that in many instances differ from the canonical import signal. In order to identify the sequences responsible for the nuclear localization, various hnRNP I portions were fused to a reporter protein (bacterial chloramphenicol acetyl transferase) and used in transient transfection assay. By this approach we identified a 60-amino-acid sequence located at the amino terminus of hnRNP I (designated NLD-I) that is both necessary and sufficient for nuclear localization. NLD-I represents a novel bipartite type of nuclear localization signal that bears no resemblance to other nuclear localization determinants so far identified in hnRNP proteins.

3T3 Cells↗
PubMed1996

APC gene mutations and allelic losses in sporadic ampullary tumours: evidence of genetic difference from tumours associated with familial adenomatous polyposis.

We explored APC gene mutations and chromosome 5q21 allelic losses (5qLOH) in 18 neoplasms of the papilla of Vater, including 6 early-stage tumours (3 adenomas, 3 carcinomas) and 12 advanced-stage cancers. Eleven PCR-amplified polymorphic sequences were used to analyse 5qLOH. APC mutations were investigated both by an in vitro APC-protein truncation test and by single-strand conformation polymorphism analysis. Mutations in the Ki-ras, N-ras and p53 genes were also assessed. We found: 5qLOH in 8 of 16 cases (50%), including 1 adenoma, 3 early- and 4 advanced-stage cancers; APC mutations in 2 adenomas and 1 advanced-stage carcinoma; Ki- or N-ras mutations in 3 adenomas and 3 advanced-stage cancers; p53 mutations in 2 early-stage and 7 advanced-stage adenocarcinomas. Our results suggest that 5qLOH, APC mutations and ras mutations are present at early stages, whereas p53 inactivation is associated with progression of malignancy in a large proportion of cases. These data indicate that sporadic ampullary tumours differ from those occurring in familial adenomatous polyposis in the frequency (17% vs. 64%) as well as in the site of APC somatic mutations, suggesting a different molecular pathogenesis in the 2 conditions.

Adenomatous Polyposis Coli↗
PubMed1996

Enhancement of HSV-DNA infectivity, in Vero and RS cells, by a modified calcium-phosphate transfection technique.

Infectivity of herpes simplex virus (HSV) DNA was assessed by employing the calcium-phosphate transfection technique described by Chen and Okayama, originally applied to increase the efficiency of plasmid transfection by N, N-bis (2-hydroxyethyl)-2-aminoethane sulfonic acid (BES). The experimental conditions and efficiency of this transfection procedure were evaluated comparing the viral progeny titers obtained by the Chen and Okayama transfection method using DNA from wild-type strains of HSV-1 and HSV-2, as well as from mutant strains, with the viral progeny obtained by the most widely used transfection technique introduced by Graham and van der Eb. Furthermore, recombinant virus production was evaluated in marker transfer and marker rescue experiments, comparing both transfection techniques, using DNA fragments cotransfected with whole viral DNA into African green monkey (Vero) or rabbit skin (RS) cells. The viral production obtained from HSV-DNA transfected cells was enhanced approximately 1000-fold when the Chen and Okayama procedure was applied.

Animals↗
PubMed1994

Analysis of the yeast NSR1 gene and protein domain comparison between Nsr1 and human hnRNP type A1.

The yeast nucleolar protein-encoding gene NSR1 was isolated by low-stringency screening of a yeast genomic library with the human heterogeneous nuclear ribonucleoprotein type A1 (hnRNP A1) cDNA probe, and was mapped to chromosome VII. RNA abundance was determined and the transcription start point and polyadenylation site were mapped. A comparison between the Nsr1 and hnRNP A1 proteins, based on homopolymer RNA binding to their structural domains in vitro, revealed a striking biochemical similarity. When the N-terminal, lysine- and arginine-rich domain of Nsr1 was removed, the truncated protein behaved similarly to hnRNP A1; furthermore, the two RRM (RNA recognition motif) domains of Nsr1 behaved in the same manner as the two RRM domains of hnRNP A1. The biochemical data, therefore, would support the hypothesis that the two RRM domains in hnRNP A1 and Nsr1 interact with RNA in a similar manner in both mammalian and yeast cells, respectively.

Base Sequence↗
PubMed1993

Relevance of DNA binding to the mechanism of anti-herpesvirus activity of benzhydrazone.

Benzhydrazone (1H-benz(f)indene-1,3(2H)-dione bis (amidino-hydrazone) (BH) is a synthetic compound with selective anti-herpesvirus activity. Its selectivity seems to stem from the inhibition of viral protein glycosylation and several hypotheses have been formulated to explain such an effect. Data presented here demonstrate that DNA binding is a prominent feature of BH. Interaction is taking place with a relatively high affinity constant and is more efficient for GC-rich viral sequences. Experiments with the cloned DNA fragments from a BH-resistant virus strain indicate that BH-DNA complex formation is drastically reduced as compared to BH-sensitive virus. The occurrence of the resistant phenotype in HEp-2 cells but not in Vero cells could be explained by differences in BH cytotoxicity. Changes in drug uptake and accumulation by cells following infection, in addition to GC preference, may also account for the degree of antiviral selectivity shown by BH.

Animals↗
PubMed1993

c-fos proto-oncogene transient transcription is negatively affected in the ELa4-2 transformed rat cell line.

To study the effects of the regulatory phosphoprotein ICP4 of the Herpes simplex virus, (HSV), a DNA tumor virus, on the induction of gene expression by the epidermal growth factor (EGF), we have constructed a cell line, ELa4-2, which constitutively expresses the a-4 gene product. The ELa4-2 cells are derived from the rat fibroblast EL2, in which EGF induces a marked c-fos and c-myc proto-oncogene transcription. Here we report that in ELa4-2 cells, the gene expression induced by EGF was negatively affected in respect to that obtained stimulating the parental EL2 cells. In particular, we studied the c-fos and c-myc proto-oncogene transcription induced by EGF. We found that in ELa4-2 cells the c-fos induction was dramatically reduced in comparison with the c-fos induction obtained in the parental EL2 cells. On the contrary, the c-myc induction by EGF was not affected by the presence of ICP4. Finally, we compared the HSV infectivity in ELa4-2 versus the EL2 cells. We showed that the virus growth capability was reduced, in the cells expressing ICP4.

Animals↗
PubMed1992

Mutational analysis of the ICP4 binding sites in the 5' transcribed noncoding domains of the herpes simplex virus 1 UL 49.5 gamma 2 gene.

A previous report (P. Mavromara-Nazos and B. Roizman, Proc. Natl. Acad. Sci. USA 86:4071-4075, 1989) demonstrated that substitution of sequences of the thymidine kinase (tk) gene, a beta gene, extending from -16 to +51 with sequences extending from -12 to +104 of the gamma 2 UL 49.5 gene in viral recombinant R3820 conferred upon the chimeric gene gamma 2 attributes in the context of the viral genome in a productive infection. The UL49.5 gene sequences extending from -179 to +104 contain four DNA binding sites for the major regulatory protein ICP4. Of these sites, two map between nucleotides +20 and +80 within the sequence which confers gamma 2 regulation upon the chimeric gene. To determine the role of these ICP4 binding sites in conferring the gamma 2 gene attributes, sequences comprising the two ICP4 binding sites were mutagenized and used to reconstruct the R3820 recombinant virus. In addition, a new recombinant virus (R8023) was constructed in which tk sequences extending from -240 to +51 were replaced with wild-type or mutated sequences contained between nucleotides -179 to +104 of the UL 49.5 gene. Vero cells infected with the recombinant viruses in the presence or absence of phosphonoacetate, a specific inhibitor of viral DNA synthesis, were then tested for accumulation of tk RNA by using an RNase protection assay. The results indicate that in the recombinant R3820, a mutation which destroyed one of the two UL49.5 ICP4 DNA binding sites significantly reduced the accumulation of tk RNA at both early and late times after infection. The effect of this mutation was less pronounced in cells infected with the R8023 virus, whose chimeric tk gene contains the two upstream UL49.5 ICP4 binding sites. None of the mutations affected the sensitivity of the chimeric genes to phosphonoacetate. The mutated site appears to be involved in the accumulation of RNA.

Animals↗
PubMed1991

Phenotypic and genotypic characterization of locus Syn 5 in herpes simplex virus 1.

Previous papers have reported that the syncytial mutant HSV-1(13)S11 carries three segregable syn mutations and exhibits its altered phenotype in four different cell lines, i.e. HEp-2, VERO, BHK and HEL both at 34 degrees C and 39 degrees C. Those studies have shown that one of three syncytial loci, designated Syn 5, is located in the Bam HI Q fragment spanning map units 0.296-0.317 of the prototype arrangement. Recombinants obtained from marker transfer experiments with donor BamHI Q fragment, have shown that locus Syn 5 is able to induce cell-to-cell fusion in VERO, BHK and HEL but not in HEp-2 cells. In this paper we have characterized the syn mutant HSV-1(13)S11 with regard to plaque morphology, synthesis of viral polypeptides and glycoproteins, thymidine kinase activity and physical map position of locus Syn 5 on the genome. Pertinent to the syn phenotype, earlier papers claimed that two different polypeptides, thymidine kinase (TK) and glycoprotein H (gH), whose genes map in BamHI Q, may be responsible for the fusion activity. Functional studies on the TK of the syn mutant HSV-1(13)S11 indicate that this polypeptide accumulates normally in infected cells and is a fully active enzyme. The other gene product, gH, has been studied with SDS-PAGE and in radioimmunoprecipitation (RIP) experiments using specific monoclonal antibodies. The results indicate that the amount of gH accumulation in the syn mutant-infected cells is greater than its parental strain. However, new marker transfer experiments described here located locus Syn 5 in 663 base pairs between SstI and EcoRI restriction endonuclease sites at the right end of the BamHI Q fragment, where TK gene overlaps in opposite orientation with UL 24 gene. Altogether these results indicate that the Syn 5 locus segregates from the gene specifying gH, to a region encompassing portions of the TK and UL 24 genes, and that the syn mutation does not affect the expression or activity of TK.

Animals↗
PubMed1991

Fine mapping and characterization of the Syn 6 locus in the herpes simplex virus type 1 genome.

The syncytial mutant of herpes simplex virus type 1 (HSV-1), HSV-1(13) S11, which carries three distinct syncytial mutations, Syn 1, Syn 5 and Syn 6, was described previously. Syn 1 maps to the BamHI L fragment, map units (m.u.) 0.707 to 0.745; Syn 5 is located within the BamHI Q fragment, m.u. 0.296 to 0.317; Syn 6 lies in the junction fragment BamHI SP, m.u. 0.81 to 0.85. Although Syn 1 of HSV-1(13) S11 seems to be homologous to that of HSV-1(MP) and other syncytial mutants, and Syn 5 has been recently characterized, Syn 6 represents a novel syncytial locus which has yet to be characterized. In this paper we report the fine mapping of the Syn 6 locus. This mutation has been mapped, by marker rescue and marker transfer experiments, to the long repeat regions (RL) at both ends of the L component of the HSV genome in a restriction endonuclease fragment of approximately 1.6 kb designated BamHI-SacI C (approximate m.u. 0.01 to 0.02 and 0.81 to 0.82). In the internal copy of RL the sequences containing the Syn 6 mutation were bounded to the left by the 5' end of the alpha gene specifying ICP0 and to the right by the gamma 1 gene encoding ICP34.5.

Animals↗
PubMed1989

Acyclovir resistance in herpes simplex virus type 1: biochemical and functional studies on the thymidine kinase of the highly resistant R100 strain.

The biochemical and functional properties of the thymidine kinase (TK) of the herpes simplex virus type 1 mutant R100, that is highly resistant to 9-(2-hydroxyethoxymethyl)guanine (acyclovir), are reported in comparison with the properties of its parental strain, wt. The mutant induced the production of a TK activity that accounted for only 10% of the wt one. This feature was not apparently related to a defective expression of the TK gene but it was rather connected to some functional characteristics of R100 enzyme. Although affinities of this enzyme for ATP and thymidine were unchanged, apparent Vmax values for thymidine were much reduced. In addition, affinities for antiviral analogues acyclovir, 9-(1,3-dihydroxymethyl)guanine (DHPG), 5-(2-bromovinyl)2'-deoxyuridine (BVdU), and 5-iodo-2'deoxycytidine (IdCyd) were drastically diminished (between 50-fold and more than 100-fold). This mutation therefore seems to affect the active site of the enzyme which is involved in the catalytic conversion of thymidine and in the binding of the analogues. The above features of HSV-1 R100 seem quite distinct from those of previously described HSV-1 resistant mutants.

Acyclovir↗
PubMed1988

A herpes simplex virus type 1 mutant resistant to benzhydrazone, an inhibitor of glycoprotein synthesis in herpesvirus-infected cells. Preliminary mapping of benzhydrazone-resistance and of a novel syncytial mutation.

Benzhydrazone (BH) is an inhibitor of glycoprotein biosynthesis. It acts selectively in Herpes simplex virus (HSV)-infected cells and does not significantly affect glycoprotein synthesis in uninfected cells and in cells infected with other viruses. Previously, we reported on a syncytial (syn) mutant, designated HSV-1(13)S11, resistant to BH, and showed that BH-resistance is encoded in the mutant virus DNA and therefore can be transferred into the genome of wild type HSVs. The present paper reports on a preliminary mapping in HSV-1(13)S11 genome of the loci which confer resistance to BH and of three distinct syn mutations present simultaneously in this mutant. Two of them were mapped in previously described syn loci localized in BamHI fragment L (map units 0.707-0.745) (locus syn 1) and BamHI fragment Q (map units 0.296-0.317) (locus syn 5). A third mutation not described before and mapping in BamHI fragment SP (c.a. map units 0.81-0.85) conferred the syn phenotype to both HEp-2 and Vero cells. This novel mutation has been designated herein locus syn 6. Transfer of BH-resistance could be achieved in cotransfection experiments involving two HSV-1(13)S11 fragments, BamHIL and BamHISP.

Animals↗
PubMed1988

Herpes simplex virus replication in the presence of DNA polymerase alpha inhibitors.

2-(p-n-butylanilino)deoxyadenosine (BuAdA), and N-2-(p-n-butylphenyl)deoxyguanosine (BuPdG), selective inhibitors of mammalian DNA polymerase alpha, were added to BHK-21(C13) cell cultures infected with herpes simplex virus type 1 (HSV-1) strain 17 syn +. Infectious virus production decreased significantly in the presence of the inhibitor at concentrations varying from 1 nM to 100 microM. BuPdG was more effective than BuAdA at all concentrations tested, while it inhibited virus yield as much as BuAdA when CVG2, a thymidine kinase deficient (TK-) HSV-1, was employed. HSV DNA synthesis, determined by quantitation of CsCl separated DNA peaks, was inhibited by each compound. BuPdG inhibited viral DNA replication more than BuAdA, while the effect on cell DNA synthesis was the same as that of BuAdA. CVG2 DNA replication was inhibited to the same level by BuAdA as by BuPdG. These results indicate that HSV DNA replication is partially dependent on cell DNA polymerase alpha activity, and that the greater effect of BuPdG on viral replication may be ascribed to its action on HSV thymidine kinase.

Animals↗