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M Giacca

Publications and source records attributed to M Giacca.

At least 109 records · Page 6Linked to original sources
PubMed1993

Searching for replication origins in mammalian DNA.

The attempts at identifying precise replication origins (ori) in mammalian DNA have been pursued mainly through physico-chemical and biochemical approaches, in view of the essential failure of the search for autonomously replicating sequences in cultured cells. These approaches involve the mapping of short stretches of nascent DNA, the identification of the regions where either leading or lagging strands switch polarity, or the localization of replication intermediates by two-dimensional gel electrophoresis. Due to the complexity of animal cell genomes, most of these studies have been performed on amplified domains and with the use of synchronization procedures. The results obtained have been controversial. In order to avoid the use of experimental procedures potentially affecting the physiological mechanism of DNA replication, we have developed a method for the localization of ori in single-copy loci in exponentially growing cells. This method entails the absolute quantification of the abundance of selected DNA fragments along a genomic region within samples of newly synthesized DNA by competitive polymerase chain reaction (PCR); the latter is immune to all the uncontrollable variables which severely affect the reproducibility of conventional PCR. The application of this method to SV40 ori-driven plasmid replication precisely identifies the known ori localization. Using the same approach, we have mapped an ori for bi-directional DNA replication in a 13.7-kb locus of human chromosome 19 encoding lamin B2.

Animals↗
PubMed1993

Interactions of USF and Ku antigen with a human DNA region containing a replication origin.

By means of a combination of ion-exchange and sequence-specific affinity chromatography techniques, we have purified to homogeneity two protein complexes binding in a human DNA region (B48) previously recognized to contain a DNA replication origin. The DNA sequence used for the protein purification (B48 binding site) contains a binding site for basic-helix-loop-helix DNA binding proteins. The first complex is composed of two polypeptides of 42- and 44-kDa; its size, heat stability, and target DNA sequence suggest that it corresponds to transcription factor USF; furthermore, the 42-kDa polypeptide is recognized by antibodies raised against 43-kDa-USF. The second complex is represented by equimolar amounts of two proteins of 72 and 87 kDa; microsequencing of the two species indicated that they correspond to the human Ku antigen. In analogy with Ku, they produce a regular pattern of footprints without an apparent sequence-specificity, and their binding can be competed by unspecific DNA provided that it contains free ends. The potential role of B48 binding site and of these cognate proteins in origin activation is discussed.

Amino Acid Sequence↗
PubMed1993

Inhibition of heterologous DNA replication by the MVMp nonstructural NS-1 protein: identification of a target sequence.

The nonstructural protein NS-1 of minute virus of mice (MVMp), an autonomous parvovirus, trans-inhibits the replication of a chimeric plasmid containing the SV40 origin of replication (ori) embedded in the MVMp genome. It appears that a 157-bp 5' proximal sequence of MVMp DNA is sufficient, in the presence of NS-1, to cause the inhibition of DNA replication driven by the SV40 ori placed on the same molecule. This effect is not dependent on the orientation of the MVMp target sequence and results from both a reduced level of utilization of SV40 ori and the blockage of progressing replication forks at the level of the target. Furthermore, replication driven by Epstein-Barr virus origin (oriP) is trans-inhibited by MVMp but this inhibition does not require the presence of parvoviral sequences in cis. On the basis of sequence homologies between EBV oriP and MVMp 5' terminal sequence, it is proposed that the direct or indirect interaction of NS-1 with parvovirus-like sequences present in heterologous viral and possibly also cellular genomes may result in an inhibition of DNA replication.

Animals↗
PubMed1993

Nested polymerase chain reaction for high-sensitivity detection of enteroviral RNA in biological samples.

A method based on nested polymerase chain reaction was developed for the detection of enteroviral genomes in biological samples. By taking advantage of the conserved 5' noncoding region of the enteroviral RNA, two sets of primers were utilized, enabling the detection either of a broad range of enteroviruses or of group B coxsackieviruses only. The sensitivity of the method is close to the detection of single molecules of viral RNA in as much as 1 mg of tissue sample. A preliminary study showed the usefulness of this technique for the analysis of endomyocardial biopsy samples from patients with idiopathic dilated cardiomyopathy and myocarditis.

Base Sequence↗
PubMed1993

In vivo footprinting analysis of constitutive and inducible protein-DNA interactions at the long terminal repeat of human immunodeficiency virus type 1.

The regulation of the rate of transcription of human immunodeficiency virus type 1 is mainly exerted through the long terminal repeat (LTR) at the 5' end of the provirus. A large number of cis-acting regulatory elements have been identified in the LTR by in vitro binding studies; the biological role of these sites within living infected cells, however, is still not clear. We have studied the interactions of nuclear proteins with the LTR in the U1 monocytic cell line by in vivo dimethylsulfate footprinting, using the ligation-mediated polymerase chain reaction technique. In this cell line, transcription of the virus, which is very low under basal conditions, is highly inducible by treatment with phorbol esters; therefore, this system is likely to represent a suitable cellular model to study viral latency. Independently of the level of viral transcription, major in vivo footprints appear at the two Sp1 sites adjacent to the enhancer, the downstream-positioned enhancer repeat, the NFAT binding site, and one of the purine-rich sites of the negative regulatory element. Upon transcriptional activation by phorbol myristate acetate, the only perturbation in the footprinting pattern is a dramatic increase in dimethylsulfate sensitivity of guanine at position -92 in the downstream enhancer repeat. This modification is correlated with the transient induction of two enhancer-binding activities, as determined by gel retardation assays. While the transcriptional rate is still increasing and the in vivo footprinting pattern is unchanged at up to 24 h postactivation, these enhancer-binding factors are considerably reduced at this time. Therefore, further levels of regulation have to be considered to explain the maintenance of the induced state.

Base Sequence↗
PubMed1992

A novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates.

A method is described for the absolute quantification by polymerase chain reaction (PCR) of nucleic acids present in low abundance. The method entails the addition to the sample of competitor DNA molecules that share the same sequence as the amplified target (including primer recognition sites), except for a 20-bp insertion in the middle, which allows easy resolution by gel electrophoresis (competitive PCR). Among the advantages of competitive PCR is that any predictable or unpredictable variable that affects amplification has the same effect on both target and competitor species and that the final ratio of amplified products reflects exactly the initial rate of targets, rendering the reaction independent of the number of amplification cycles. An easy and reliable method for the construction and quantification of competitive templates obtained as recombinant PCR products was developed. The technique was used for the absolute quantification of human genomic DNA with primers from a single copy, subtelomeric region of chromosome 19.

Base Sequence↗
PubMed1992

Lack of detectable human T-cell lymphotropic virus type I sequences in samples from multiple sclerosis patients.

Recently, inconclusive results have followed the early data on the possible association between multiple sclerosis (MS) and human T-cell lymphotropic virus type I (HTLV-I) infection. For this reason, we examined this hypothesis using the polymerase chain reaction (PCR) to study samples of differing origin from Italian MS patients. In particular, we developed a systematic analysis of paraffin-embedded brain white matter from histologically defined lesions of 14 MS patients using PCR and primer sets specific for HTLV-I sequences; additionally, cerebrospinal fluids (CSFs) from 12 patients and peripheral blood mononuclear cells (PBMCs) from subjects at the early and late phase of the disease were investigated for free HTLV-I virions and specific proviral sequences, respectively. In agreement with some groups who reported lack of HTLV-I sequences in PBMCs of MS patients but in clear contrast with others, we failed to detect specific viral sequences using this broad approach.

Base Sequence↗
PubMed1992

A human DNA replication origin: localization and transcriptional characterization.

A single-copy 13.7 kb human DNA region (L30E) located on Ch. 19 p13.3 contains an origin of DNA replication in myeloid HL-60 cells. The origin was localized, by means of quantitative PCR within approximately 3000 bp, in a highly transcribed region containing at least two closely spaced genes with the same polarity of transcription, one encoding lamin B2 and the other an unidentified protein. The origin region overlaps an undermethylated "CpG island" at the 5'-end of the second transcription unit. A binding site (CACGTG) for basic helix-loop-helix (bHLH) DNA binding proteins such as USF/MLTF or MYC-MAX was located by DNase I footprinting analysis in the promoter of the second gene. DMSO differentiation of HL-60 cells, that completely shuts off replication, also drastically reduces the transcription of L30E region. On the other hand such treatment does not modify the methylation pattern of the CpG island and does not abolish the DNase I protection of the bHLH binding site.

Base Sequence↗
PubMed1992

A human binding site for transcription factor USF/MLTF mimics the negative regulatory element of human immunodeficiency virus type 1.

Transcriptional regulation of the proviral form of the human immunodeficiency virus type 1 (HIV-1) is exerted by its 5' long terminal repeat (LTR), which contains recognition sites for several cell factors. By gel retardation and DNase I footprinting experiments we have identified a binding site for a human nuclear protein between nucleotides -152 to -174 upstream of transcription start site, in a region previously recognized as a negative regulator of transcription (negative regulatory element, NRE). The recognized sequence contains the dyad symmetry element CACGTG, which represents a binding motif, very conserved through evolution, present in a putative human DNA replication origin (pB48), in the upstream element of the major late promoter (MLP-UE) of adenovirus, and, as transcriptional element, upstream of many eukaryotic genes. Common binding activities exist in human nuclear extracts for pB48, MLP-UE and the HIV-1 LTR; at least three protein species recognize the LTR sequence, of 44 (corresponding to transcription factor USF/MLTF), 70, and 110 kDa, respectively. Chloramphenicol acetyltransferase assays suggest that the USF/MLTF binding site located in the HIV-1 LTR acts as a negative regulator of transcription, and that it contributes to the overall negative function exerted by the NRE. An oligonucleotide corresponding to another characterized human USF/MLTF binding site can functionally replace part of the activity of the NRE. This negative function is exerted both in presence or absence of tat transactivation, in different cell lines, and after PMA stimulation.

Adenoviruses, Human↗
PubMed1992

Absolute quantitation of viremia in human immunodeficiency virus infection by competitive reverse transcription and polymerase chain reaction.

A competitive polymerase chain reaction (PCR)-based assay for the quantitative detection of human immunodeficiency virus type 1 (HIV-1) viremia was developed and optimized. This method consists of the reverse transcription and subsequent amplification in the same tube of two similar RNA templates, the wild-type template to be quantified and a known amount of the internally deleted synthetic template, both with identical primer recognition sites. The same strategy also proved to be useful in the quantitative assay of HIV-1-specific cellular transcripts and proviral DNA sequences from peripheral blood mononuclear cells by using competitor DNA. The method might be of interest in the study of the precise level of HIV-1 activity during the different clinical phases of the infection and in the simple, fast, and methodologically correct molecular investigation of patients treated with specific antiviral compounds.

Base Sequence↗
PubMed1992

Molecular profile of human immunodeficiency virus type 1 infection in symptomless patients and in patients with AIDS.

Recent molecular evidence indicates that active human immunodeficiency virus type 1 (HIV-1) infection is detectable in both symptomless and symptomatic infected patients. For this main reason, it has been pointed out that precise quantitative analysis of viral activity in vivo is necessary, firstly, for the pathogenetic investigation of the steps relevant to infection progression and, secondly, for better clinical management of HIV-1-infected patients. In this study, the presence of HIV-1 genomic RNA in plasma samples, specific HIV-1 transcripts in peripheral blood mononuclear cells, and proviral DNA sequences were assayed for 33 HIV-1-infected patients (including symptomless and symptomatic subjects) by using a competitive polymerase chain reaction method that allows quantitation of the RNA/DNA target sequences. The quantitative results obtained confirm that transcription of HIV-1 structural genes and complete viral replication occur in all the HIV-1-infected patients independently of the clinical stage. However, although sharp individual differences were detected, a high degree of correlation of the molecular parameters studied with both disease progression and a decrease in the number of CD4+ T lymphocytes was documented. Interestingly, despite the increasing viremia level associated with infection progression, the mean transcriptional activity of individual infected cells was found to be only moderately greater in AIDS patients than in asymptomatic infected subjects. In addition, it was noted that quantitation of HIV-1 genomic RNA in plasma samples and quantitation of specific HIV-1 transcripts in peripheral blood mononuclear cells appear to be more reliable and sensitive markers of viral activity than quantitative analysis of proviral HIV-1 sequences in peripheral lymphocytes.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Probing protein-DNA interactions at the long terminal repeat of human immunodeficiency virus type 1 by in vivo footprinting.

We have analyzed protein-DNA interactions at the long terminal repeat of human immunodeficiency virus type 1 in a productively infected T-cell line by in vivo dimethyl sulfate footprinting. Major footprints are evident at the basal promoter and enhancer elements. In particular, proteins appear to occupy the TATA box, the Sp1 sites, and the two repeats of the enhancer region. In the negative regulatory element, protections are detected over the USF/MLTF and NFAT-1 sites. Furthermore, two previously unrecognized sites, from nucleotides -260 to -275 and from nucleotides -205 to -216, respectively, appear to be involved in protein-DNA interactions. These two sites are purine rich and share a common sequence motif.

Base Sequence↗
PubMed1992

The gene for a novel human lamin maps at a highly transcribed locus of chromosome 19 which replicates at the onset of S-phase.

A previously described human DNA fragment which is replicated early in S-phase of HL-60 cell DNA (C. Tribioli, G. Biamonti, M. Giacca, M. Colonna, S. Riva, and A. Falaschi, Nucleic Acids Res. 15:10211-10232, 1987) was used to screen a genomic library in lambda Ch28. A clone which contained a 13.7-kb insert (L30E) found to code for several transcripts was isolated. The transcription of L30E DNA exhibited a complex pattern and a tissue-specific and proliferation-dependent type of regulation. The data were consistent with two tandemly arranged transcription units, the 3' end of one separated from the 5' end of the other by a sequence of about 600 bp containing an active promoter. The isolation and sequencing of L30E-specific cDNAs permitted identification of two genes, one of which encoded a B-type human lamin (analogous to mouse lamin B2). L30E DNA was mapped by in situ hybridization at the G-negative subtelomeric band p13.3 of chromosome 19. Interestingly, in synchronized HL-60 cells, L30E DNA is replicated in the first minute of S-phase. Replication of the lamin gene early in S-phase may reflect a coupling between early replication and transcription of genes for S-phase-specific proteins such as lamins.

Amino Acid Sequence↗
PubMed1992

[The pathogenesis of dilated cardiomyopathy: current progress].

The pathogenesis of dilated cardiomyopathy (DCM) is still unknown; however, some factors that seem to play an important role in the development of the disease have recently been identified: they are enteroviral infections, immune mechanisms and genetic factors. Enteroviral infection (particularly due to Coxsackie virus B) has long been suspected to be the cause of myocarditis and subsequent DCM. However, only recent techniques of genetic engineering have been able to demonstrate the presence of enteroviral RNA in endomyocardial biopsy of patients with DCM. The role of the viral particles contained in the myocardium is still undetermined. Changes in the immune system concerning cell-mediated and humoral immunity have been recently detected. It has been suggested that an autoimmune process could be the actual cause of DCM in some patients, rather than the consequence. The immune system is strictly related to the major histocompatibility complex. As in some autoimmune diseases, a relationship between DCM and HLA class II phenotype has been found: particularly the DR4 antigen seems to be associated with a high risk of disease. Besides immunogenetic factors, other genetic factors seem to play a role in the pathogenesis of DCM. In 6-8% of cases a familial history of cardiomyopathy has been observed. In clinical studies on familial DCM different phenotypes have been shown, suggesting that different genetic mechanisms are involved in the pathogenesis of the disease. At least two main mechanisms can be hypothesized: the transmission of "predisposing" factors or a defect in proteins essential for the cardiac muscle cell function. Viral agents, autoimmune reactions, immunogenetic and genetic factors seem to cause myocardial damage individually or with complex interactions: the research should be devoted to these topics in the future.

Antibody Formation↗
PubMed1991

Detection of human immunodeficiency virus type 1 genomic RNA in plasma samples by reverse-transcription polymerase chain reaction.

An application of the polymerase chain reaction (PCR) to the direct detection of human immunodeficiency virus type 1 (HIV-1) viremia is described. The amplification of specific HIV-1 sequences of gag and env viral genes was carried out after the reverse-transcription of plasma samples (plasma RT-PCR) from seropositive subjects. The assay is faster and cheaper than detection of specific HIV-1 transcripts from peripheral blood mononuclear cells by RT-PCR. The data suggest that HIV-1 viremia is detectable by plasma RT-PCR in a large proportion of seropositive individuals.

Genes, env↗
PubMed1991

Detection of human immunodeficiency virus type 1 transcripts in peripheral blood lymphocytes by the polymerase chain reaction.

A simplified application of the polymerase chain reaction (PCR) to the routine detection of human immunodeficiency virus type 1 (HIV-1) transcripts from peripheral lymphocytes of infected subjects is described. This technique is simpler than previously described assays and was shown to be highly sensitive after ethidium bromide staining of polyacrylamide gel electrophoresis of amplified material. The method can be used for the virologic evaluation of HIV-1-infected subjects, thus allowing early identification of seropositive patients with signs of active infection.

Cell Line↗