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P Donini

Publications and source records attributed to P Donini.

At least 19 recordsLinked to original sources
PubMed2001

Molecular and cytological analysis of a 5.5 Mb minichromosome.

Mammalian artificial chromosomes (MACs) provide a new tool for the improvement of our knowledge of chromosome structure and function. Moreover, they constitute an alternative and potentially powerful tool for gene delivery both in cultured cells and for the production of transgenic animals. In the present work we describe the molecular structure of MC1, a human minichromosome derived from chromosome 1. By means of restriction and hybridization analysis, satellite-PCR, in situ hybridization on highly extended chromatin fibres, and indirect immunofluorescence, we have established that: (i) MC1 has a size of 5.5 Mb; (ii) it consists of 1.1 Mb alphoid, 3.5 Mb Sat2 DNA, and telomeric and subtelomeric sequences at both ends; (iii) it contains an unusual region of interspersed Sat2 and alphoid DNAs at the junction of the alphoid and the Sat2 blocks; and (iv) the two alphoid blocks and the Sat2-alphoid region bind centromeric proteins suggesting that they participate in the formation of a functional kinetochore.

Animals↗
PubMed1999

Use of a human minichromosome as a cloning and expression vector for mammalian cells.

A natural human minichromosome (MC1) derived from human chromosome 1 was shown to be linear and to have a size of 5.5 Mb. Human IL-2 cDNA and the neo gene were co-transfected into a MC1-containing human-CHO hybrid cell line. Integration of the foreign genes was directed to the pericentromeric region of MC1 by co-transfection of chromosome 1-specific satellite 2 DNA. A number of G418-resistant transfectants were obtained and expression of IL-2 was determined. FISH analysis demonstrated co-localization in the minichromosome of the IL-2 gene and of the satellite 2 DNA. An IL-2-producing clone was used in cell fusion experiments with IL-2-dependent murine CTLL cells to generate CTLL-human hybrids containing the modified minichromosome (MC1- IL2 ). The hybrids were able to grow in medium lacking IL-2 for 17 mean population doublings (MPD), indicating that expression of the cytokine was sufficient to relieve the IL-2 dependence of CTLL proliferation. Endogenous IL-2 production delayed the onset of apoptosis in the IL-2-dependent CTLL cells. Mitotic stability was shown to be 100% in the human-CHO hybrids and 97% per MPD in CTLL cells. These results demonstrate that a natural human minichromosome can be utilized as a cloning and expression vector for mammalian cells and that the MC1 minichromosome can be engineered to deliver IL-2 to two types of cells, fibroblasts and lymphocytes.

Animals↗
PubMed1999

Helicobacter pylori: a eubacterium lacking the stringent response.

Accumulation of 16S rRNA and production of guanosine polyphosphates (pppGpp and ppGpp) were studied during amino acid starvation in three wild-type strains of Helicobacter pylori. All strains exhibit a relaxed phenotype with respect to accumulation of 16S rRNA. This constitutes the first example of a wild-type eubacterium showing a relaxed phenotype. The guanosine polyphosphate levels do not rise as a result of amino acid starvation, as expected for relaxed organisms. However, in both growing and starved cells, basal levels of the two polyphosphates appeared to be present, demonstrating that the enzymatic machinery for guanosine polyphosphate production is present in this organism. These findings are discussed within the framework of the hypothesis that stringent control is a physiological control mechanism more important for the fitness of prokaryotes growing in the general environment than for those that inhabit protected niches.

Amino Acids↗
PubMed1997

Mammalian artificial chromosomes--vectors for somatic gene therapy.

Mammalian artificial chromosomes might prove to be useful vectors for somatic gene therapy. The functional elements of such an artificial chromosome are telomeres, a centromere and a replication origin. Recent progress in the characterization of these functional elements of the eukaryotic chromosome will be described. Attempts to construct artificial chromosomes for mammalian cells and their use for somatic gene therapy are discussed.

Animals↗
PubMed1997

Yeast linear plasmids with T2AG3 telomeres: TEL+CEN antagonism and genetic and molecular stability.

A linear plasmid containing ARS1, CEN4, and 48 bp of vertebrate (T2AG3) telomeric sequences at each end was used to transform Saccharomyces cerevisiae. Only circular plasmids that had lost the centromere and had retained the T2AG3 sequences were obtained, indicating that the vertebrate T2AG3 sequences and the yeast CEN4 could not be simultaneously present in this vector. This hypothesis was verified by removing the CEN4 sequence from the construct. In fact, the resulting transformants contained two classes of efficiently replicating linear plasmids: one of the expected size and one about twice as large. During subsequent growth, plasmids of the former, but not latter, class were subjected to concatemer formation. This can best be explained by recombination events involving the T2AG3 sequences at the ends of the molecule, since very similar centric and acentric linear plasmids bearing Tetrahymena telomeric ends replicated faithfully.

Centromere↗
PubMed1995

Ligation of nonmatching DNA molecule ends.

T4 DNA ligase can promote the in vitro ligation of blunt DNA ends to ends bearing a 2-nucleotide single-stranded protrusion. This was shown by digestion of plasmids pBR322 and pSP71 with the appropriate restriction enzymes followed by recircularization of the plasmids and transformation of Escherichia coli. It could be ruled out that such nonmatching ligations are due to the presence of contaminating nucleases. The efficiency of ligation is of the same order of magnitude as that obtained with blunt end ligations. The interaction of a number of different combinations of blunt and sticky ends, the latter bearing both 3' and 5' protrusions, was investigated. Ligation of nonmatching ends was shown to take place in all cases. Several ligation junctions were sequenced, showing that during the ligation process the 2-nucleotide protrusion is trimmed away. In two instances the ligation event was accompanied by the specific loss of either 3 or 15 nucleotide pairs as well as the protrusion. An intermolecular ligation involving nonmatching ends was also performed, demonstrating that this form of ligation can be usefully employed in molecular cloning experiments.

Base Sequence↗
PubMed1995

Lack of production of (p)ppGpp in Halobacterium volcanii under conditions that are effective in the eubacteria.

The stringent halobacterial strain Haloferax volcanii was subjected to a set of physiological conditions different from amino acid starvation that are known to cause production of guanosine polyphosphates [(p)pp Gpp] in eubacteria via the relA-independent (spoT) pathway. The conditions used were temperature upshift, treatment with cyanide, and total starvation. Under none of these conditions were detectable levels of (p)ppGpp observed. This result, in conjunction with our previous finding that (p)ppGpp synthesis does not occur under amino acid starvation, leads to the conclusion that in halobacteria both growth rate control and stringency are probably governed by mechanisms that operate in the absence of ppGpp. During exponential growth, a low level of phosphorylated compounds with electrophoretic mobilities similar, but not identical, to that of (p)ppGpp were observed. The intracellular concentration of these compounds increased considerably during the stationary phase of growth and with all of the treatments used. The compounds were identified as short-chain polyphosphates identical to those found under similar conditions in Saccharomyces cerevisiae.

Culture Media↗
PubMed1994

In vitro identification of a protein of Saccharomyces cerevisiae that interacts specifically with the G-rich DNA strand of the telomere.

The telomeres of Saccharomyces cerevisiae consist of a repeated G2-3T(GT)1-6 DNA sequence that forms a complex with proteins. To date only the RAP1 protein has been shown to bind to the simple sequences in yeast telomeric DNA, as well as to non-telomeric regulatory sites. We have used synthetic oligodeoxyribonucleotides, both double- and single-stranded, to identify specific yeast telomeric proteins in a partially purified yeast extract. Using the gel shift assay, we detected a binding activity that is stable at high ionic strength and that recognizes specifically the G-rich protrusion of a double-stranded synthetic yeast telomere, as well as the G-rich single strand. This is the first evidence of a purely telomeric protein in that it binds to the single-stranded telomeric protrusion of the yeast chromosome.

DNA, Fungal↗
PubMed1993

Subtelomeric as well as telomeric sequences are lost from chromosomes in proliferating B lymphocytes.

B lymphocytes purified from peripheral blood can be normally cultured in vitro for only one doubling. They can undergo an unlimited number of cell divisions after transformation with a DNA tumor virus such as the Epstein-Barr virus. We have shown that the terminal restriction fragments of virus transformed B lymphocytes are shortened in the course of proliferation and that this process is accompanied by structural modifications. We have identified the sequences that are lost during the shortening process by hybridization to the canonical human telomeric simple repeat TTAGGG, to other simple sequences that are found at the ends of human chromosomes, and to a human subtelomeric sequence. We have observed that by 20 doublings over half the TTAGGG sequences, but few or no TGAGGG sequences, are lost from the TRFs. The subtelomeric sequence was removed from most of the TRFs on which it was present. The implications that these observations have on the problems of cell senescence and oncology are discussed.

B-Lymphocytes↗
PubMed1993

Stringency and relaxation among the halobacteria.

Accumulation of stable RNA and production of guanosine polyphosphates (ppGpp and pppGpp) were studied during amino acid starvation in four species of halobacteria. In two of the four species, stable RNA was under stringent control, whereas one of the remaining two species was relaxed and the other gave an intermediate phenotype. The stringent reaction was reversed by anisomycin, an effect analogous to the chloroamphenicol-induced reversal of stringency in the eubacteria. During the stringent response, neither ppGpp nor pppGpp accumulation took place during starvation. In both growing and starved cells a very low basal level of the two polyphosphates appeared to be present. In the stringent species the intracellular concentration of GTP did not diminish but actually increased during the course of the stringent response. These data demonstrate that (i) wild-type halobacteria can have either the stringent or the relaxed phenotype (all wild-type eubacteria tested have been shown to be stringent); (ii) stringency in the halobacteria is dependent on the deaminoacylation of tRNA, as in the eubacteria; and (iii) in the halobacteria, ppGpp is not an effector of stringent control over stable-RNA synthesis.

Bacterial Proteins↗
PubMed1991

On the level of plasmid-bearing cells in transformed cultures of Saccharomyces cerevisiae.

LC1, a YIP5-derived plasmid containing a human DNA fragment with ARS activity in yeast, has been used to study the replication of ARS plasmids in Saccharomyces cerevisiae. ARS plasmids carried in yeast hosts are normally mitotically unstable. In transformed cultures the fraction of cells that contain plasmid, measured by plating on selective media, is lower than would be expected from measured rates of plasmid loss. In the case of S. cerevisiae carrying either the plasmid LC1 or YRP17, the assay yields values of the order of 10-20% or 30-50% respectively. We have found that by doing a double nutritional upshift that involves conditioned medium and casamino acids, a population of cells can be defined that carry plasmid but are unable to grow on media that select for the plasmid marker. Thus the total fraction of cells that can be shown to contain plasmid increases to greater than 70%. To distinguish between the inability of plasmid to replicate in these cells and lack of expression of the selectable gene, cultures grown from single cells were analysed for the presence of plasmid DNA. In a substantial fraction of the population, plasmid DNA could be detected only by polymerase chain reaction and not by standard blotting and hybridization. These results suggest that plasmid is unable to replicate in these cells. Growth kinetics experiments with transformed cultures are consistent with the notion that only a small fraction of the cells contains plasmid capable of replication upon dilution into selective medium. Possible explanations for the phenomena observed are discussed.

Culture Media↗
PubMed1990

Cloning a fragment from the telomere of the long arm of human chromosome 9 in a YAC vector.

The construction of a yeast artificial chromosome containing a human DNA insert is reported. This molecule of about 200 kb behaves as a native yeast chromosome since it has a very high mitotic stability and is present in the yeast transformant clone at a copy number similar to that of the resident chromosomes. Hybridization with the TTAGGG sequence demonstrates that this chromosome contains human telomeric sequences. In situ hybridization of the biotin-labelled artificial chromosome to metaphase human chromosomes shows that the insert occupies a telomeric position on the long arm of chromosome 9. Since the fragment was cloned as an EcoRI insert and not as a telomere, it is situated medially to the telomeric sequences and harbours telomere-associated sequences, that have been shown to contain the TTAGGG sequence. The fragment represents the end of the genetic map of chromosome 9 and thus can be used to characterize the sequence and the structure of the chromosomal region that runs from the end of the chromosome to the first gene.

Base Sequence↗
PubMed1990

Functional telomere formation in yeast using synthetic C4A2 sequences.

A yeast artificial chromosome (YAC) was constructed with a native autonomous replicating sequence (ARS) flanked telomere at one end and a 50-bp synthetic oligonucleotide of C4A2 repeats at the other. This was done in order to determine whether the presence of the flanking ARS sequence is required for telomere function. This construct was introduced into two different yeast strains: one mutated in the recombination function RAD52 and the other wild type for this gene. Both strains gave rise to autonomously replicating artificial chromosomes. The molecules in the RAD52 strain were rearranged dimers terminating at both ends with Tetrahymena telomeres, whereas in the rad52 strain two classes of YACs were found: rearranged dimers and elements bearing an ARS-free telomere. The presence of the latter class of molecules confirmed the finding of Wellinger and Zakian (1989, Proc. Natl. Acad. Sci. USA 86, 973-977) that the flanking ARS is not required for telomere function. Furthermore, in this class of molecules the ARS-free telomeric end was shortened as a result of deletions that removed some distal pBR322 sequences and some C4A2 repeats. The size of the resulting YACs ranged from 7.7 to 9 kb, considerably below the size threshold found by Zakian et al. (1986, Mol. Cell. Biol. 6, 925-932) for CEN4 artificial plasmids. An explanation for the structural instability of the ARS-free end of the YACs is suggested.

Adenosine Triphosphate↗
PubMed1989

Relationship between guanosine tetraphosphate and accuracy of translation in Salmonella typhimurium.

In bacteria a high level of mistranslation is observed in amino acid starved rel-, but not rel+, strains, and mistranslation can be studied qualitatively by means of "stuttering" experiments in two-dimensional protein gels. It has been suggested that the low level of mistranslation that occurs in rel+ strains is assured by guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a nucleotide whose intracellular concentration greatly increases in rel+ cells under amino acid starvation. In the present study the relationship between level of ppGpp and mistranslation was analyzed by performing stuttering experiments in amino acid starved bacteria that contained either high or low levels of ppGpp. Three strains of Salmonella typhimurium were used in these experiments: a relA+ hisT+ strain (TA997), a relA+ hisT strain (TA1001), and a relA hisT strain (PD2). These strains were first characterized with respect to macromolecular syntheses and ppGpp levels under exponential growth and under amino acid starvation. Both rel+ strains exhibited stringent control over RNA synthesis. ppGpp accumulated to high levels when TA997 was starved for either of three amino acids. Starvation of TA1001 for histidine did not cause accumulation of ppGpp, whereas starvation for lysine and arginine produced high levels of ppGpp. Extracts from the three strains, obtained either under exponential growth or under amino acid starvation, were then subjected to two-dimensional electrophoretic anaylsis: mistranslation was observed whenever ppGpp was absent. In particular, starvation of TA1001 for histidine resulted in high mistranslation frequencies, while under lysine and arginine starvation mistranslation was undetectable, regardless of whether the cells were rel+ or rel-.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1988

Inaccurate protein synthesis in a mutant of Salmonella typhimurium defective in transfer RNA pseudouridylation.

Protein synthesis was studied comparatively in a wild-type strain of Salmonella typhimurium and in hisT mutant cells defective in the pseudouridylation of transfer RNA. From a quantitative point of view, no significant differences between the two types of strain was observed when measuring the rate of protein synthesis during either exponential growth or starvation for histidine. In contrast, the qualitative analysis of proteins by two-dimensional gel electrophoresis showed that histidine-starved hisT cells mistranslate the genetic program at a higher frequency than exponentially growing hisT cells or either starved or unstarved hisT+ cells.

Bacterial Proteins↗
PubMed1985

Transformation of Saccharomyces cerevisiae and Schizosaccharomyces pombe with linear plasmids containing 2 micron sequences.

Linear plasmids were constructed by adding telomeres prepared from Tetrahymena pyriformis rDNA to a circular hybrid Escherichia coli-yeast vector and transforming Saccharomyces cerevisiae. The parental vector contained the entire 2 mu yeast circle and the LEU gene from S. cerevisiae. Three transformed clones were shown to contain linear plasmids which were characterized by restriction analysis and shown to be rearranged versions of the desired linear plasmids. The plasmids obtained were imperfect palindromes: part of the parental vector was present in duplicated form, part as unique sequences and part was absent. The sequences that had been lost included a large portion of the 2 mu circle. The telomeres were approximately 450 bp longer than those of T. pyriformis. DNA prepared from transformed S. cerevisiae clones was used to transform Schizosaccharomyces pombe. The transformed S. pombe clones contained linear plasmids identical in structure to their linear parents in S. cerevisiae. No structural re-arrangements or integration into S. pombe was observed. Little or no telomere growth had occurred after transfer from S. cerevisiae to S. pombe. A model is proposed to explain the genesis of the plasmids.

Ascomycota↗