PubMed Health⌕ Search

Biomedical subjects

Z Larin

Publications and source records attributed to Z Larin.

At least 19 recordsLinked to original sources
PubMed2001

Functional complementation of a genetic deficiency with human artificial chromosomes.

We have shown functional complementation of a genetic deficiency in human cultured cells, using artificial chromosomes derived from cloned human genomic fragments. A 404-kb human-artificial-chromosome (HAC) vector, consisting of 220 kb of alphoid DNA from the centromere of chromosome 17, human telomeres, and the hypoxanthine guanine phosphoribosyltransferase (HPRT) genomic locus, was transferred to HPRT-deficient HT1080 fibrosarcoma cells. We generated several cell lines with low-copy-number, megabase-sized HACs containing a functional centromere and one or possibly several copies of the HPRT1 gene complementing the metabolic deficiency. The HACs consisted of alternating alphoid and nonalphoid DNA segments derived only from the input DNA (within the sensitivity limits of FISH detection), and the largest continuous alphoid segment was 158-250 kb. The study of both the structure and mitotic stability of these HACs offers insights into the mechanisms of centromere formation in synthetic chromosomes and will further the development of this human-gene-transfer technology.

Blotting, Southern↗
PubMed2000

The assembly of large BACs by in vivo recombination.

We have developed a method for recombining bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) containing large genomic DNA fragments into a single vector using the Cre-lox recombination system from bacteriophage P1 in vivo. This overcomes the limitations of in vitro methods for generating large constructs based on restriction digestion, ligation, and transformation of DNA into Escherichia coli cells. We used the method to construct a human artificial chromosome vector of 404 kb encompassing long tracts of alpha satellite DNA, telomeric sequences, and the human hypoxanthine phosphoribosyltransferase gene. The specificity of Cre recombinase for loxP sites minimizes the possibility of intramolecular rearrangements, unlike previous techniques using general homologous recombination in E. coli, and makes our method compatible with the presence of large arrays of repeated sequences in cloned DNA. This methodology may also be applied to retrofitting PACs or BACs with markers and functional sequences.

Base Sequence↗
PubMed2000

Stable integration of large (>100 kb) PAC constructs in HaCaT keratinocytes using an integrin-targeting peptide delivery system.

Transfer of large DNA constructs in gene therapy studies is being recognised for its importance in maintaining the natural genomic environment of the gene of interest and providing tissue-specific regulation and control. However, methods used to deliver such constructs have been poorly studied. We used a receptor-mediated, integrin-targeting transfection system enhanced by liposomes, to deliver a 110 kb PAC (P1-based artificial chromosome) to HaCaT keratinocytes. The PAC contained the collagen VII locus, an EGFP (enhanced green fluorescent protein) reporter gene and the puromycin resistance gene (pac) to allow selection of stably transfected cells. Analysis of puromycin resistant and EGFP-expressing colonies by Western blot showed that collagen VII production increased dramatically after transfection, indicating successful transfer of a large fully functional genomic locus. Fluorescent in situ hybridisation (FISH) and Southern blot analysis revealed that the PAC had integrated as at least one copy per cell. EGFP expression has persisted for 35 weeks, suggesting stable transgene expression. We conclude that the integrin-targeting peptide method of gene delivery is an effective means of stably delivering large DNA constructs to human keratinocytes and could be of benefit for genomic gene therapy approaches.

Anti-Bacterial Agents↗
PubMed1999

11q23.1 and 11q25-qter YACs suppress tumour growth in vivo.

Frequent allelic deletion at chromosome 11q22-q23.1 has been described in breast cancer and a number of other malignancies, suggesting putative tumour suppressor gene(s) within the approximately 8 Mb deleted region. In addition, we recently described another locus, at the 11q25-qter region, frequently deleted in breast cancer, suggesting additional tumour suppressor gene(s) in this approximately 2 Mb deleted region. An 11q YAC contig was accessed and three YACs, one containing the candidate gene ATM at 11q23.1, and two contiguous YACs (overlapping for approximately 400-600 kb) overlying most of the 11q25 deleted region, were retrofitted with a G418 resistance marker and transfected into murine A9 fibrosarcoma cells. Selected A9 transfectant clones (and control untransfected and 'irrelevant' alphoid YAC transfectant A9 clones) were assayed for in vivo tumorigenicity in athymic female Balb c-nu/nu mice. All the 11q YAC transfectant clones demonstrated significant tumour suppression compared to the control untransfected and 'irrelevant' YAC transfected A9 cells. These results define two discrete tumour suppressor loci on chromosome 11q by functional complementation, one to a approximately 1.2 Mb region on 11q23.1 (containing the ATM locus) and another to a approximately 400-600 kb subterminal region on 11q25-qter.

Animals↗
PubMed1999

Transfer of YACs up to 2.3 Mb intact into human cells with polyethylenimine.

The transfer of large YAC DNA into human cells is a laborious procedure. High quality pulsed field gel purified DNA is required, which is easily sheared during manipulation before transfection or degraded in the endosome of the cell following transfection. NaCl and polyamines compact and prevent DNA from shearing, but may not consistently protect DNA after transfection. We investigated if other polycations such as poly-L-lysine (PLL) and polyethylenimine (PEI) could condense and protect large YAC DNA (up to 2.3 Mb) from being degraded after lipofection. DNA condensation was monitored by a gel retardation assay, and atomic force microscopy (AFM). DNA was retarded in the gel when complexed with high concentrations of PLL and PEI, indicating that DNA had condensed. However, AFM images of PLL-DNA complexes showed aggregates of DNA molecules resulting from incomplete condensation, whereas PEI-DNA complexes produced condensed particles approximately 30-60 nm. Exogenous PLL-DNA remained intact in 36% of positive clones after lipofection, whereas PEI-DNA was intact in 100% of positive clones. PEI is a better condensing reagent than PLL, protecting DNA from shearing and endosomal degradation, and assists in delivering YACs up to 2.3 Mb intact into human cells.

Cell Line↗
PubMed1997

Generation of large insert yeast artificial chromosome libraries.

The development of YAC cloning technology has directly enhanced the relationship among genetic, physical, and functional mapping of genomes. Because of their large size, YACs have enabled the rapid construction of physical maps by ordered clone mapping and contig building, and they complement other molecular approaches for mapping complex genomes. Large insert libraries are constructed by size fractionating large DNA embedded in agarose and protecting DNA from degradation with polyamines.

Chromosomes, Artificial, Yeast↗
PubMed1996

A method for linking yeast artificial chromosomes.

A method for linking any standard yeast artificial chromosomes (YAC) is described. YACs are introduced into the same cell and joined by mitotic recombination between the vector arms and the homologous sequence in a linking vector; several YACs can be recombined sequentially. The linking vectors also contain the beta-galactosidase gene as an expression reporter in mammalian cells.

Cell Line↗
PubMed1996

Physical analysis of the region deleted in the tw18 allele of the mouse tcl-4 complementation group.

We have generated a YAC contig of at least 3.3 Mb from the proximal region of In(17)4 of mouse chromosome 17. This region corresponds to DNA lost in the gastrulation mutant tw18, which belongs to the tcl-4 complementation group. Our most proximal and distal probes lie within the deletion-3.3 Mb apart-indicating that we have not cloned the entire region. The deleted region is contained in a genetic interval of less than 1 cM, suggesting that some suppression of recombination must occur.

Alleles↗
PubMed1996

Analysis of extrachromosomal structures containing human centromeric alphoid satellite DNA sequences in mouse cells.

Yeast artificial chromosomes (YACs) spanning the centromeric region of the human Y chromosome were introduced into mouse LA-9 cells by spheroplast fusion in order to determine whether they would form mammalian artificial chromosomes. In about 50% of the cell lines generated, the YAC DNA was associated with circular extrachromosomal structures. These episomes were only present in a proportion of the cells, usually at high copy number, and were lost rapidly in the absence of selection. These observations suggest that, despite the presence of centromeric sequences, the structures were not segregating efficiently and thus were not forming artificial chromosomes. However, extrachromosomal structures containing alphoid DNA appeared cytogenetically smaller than those lacking it, as long as yeast DNA was also absent. This suggests that alphoid DNA can generate the condensed chromatin structure at the centromere.

Animals↗
PubMed1995

MHC class I gene organization in > 1.5-Mb YAC contigs from the H2-M region.

Sixteen yeast artificial chromosome (YAC) clones have been mapped to the H2-M region at the distal end of the mouse major histocompatibility complex (MHC) on chromosome 17. Analysis of the YACs with single- and multicopy probes yielded a proximal contig spanning a minimum of 800 kb and a distal contig of 700 kb. A probe for the conserved fourth exon of MHC class I genes detected 19 restriction fragments, including 6 of the 8 previously characterized H2-M class I genes, in the proximal contig. This contig spans the gap from the M to the T region and includes the T1 gene. By contrast, only two class I genes, M2 and M3, were found in the distal contig. These two genes, which are both expressed, may mark the end of the MHC. The order among nine class I genes and seven other markers was determined in the cloned DNA from the centromere as T1, Tu32A, (M1-M7-M8), Tu32B, B30, M6, M4, M5, Mog, Tu42A parallel M2, Leh525, M3, Tu42B, where the orientation with respect to the centromere is unknown for M1-M7-M8.

Animals↗
PubMed1994

Fluorescence in situ hybridisation of multiple probes on a single microscope slide.

We report a method to analyse multiple samples by fluorescence in situ hybridisation on a single glass microscope slide. Wells were formed in which independent hybridisation reactions could proceed by sealing a silicon rubber gasket to the slide. In the largest format tested, different probes were hybridised simultaneously by applying them directly from a 96-well microtitre dish which was inverted on a glass plate. This technique will increase the rate of analysis of multiple probes against a standard set of chromosomes and could also be used to analyse different karyotypes using a panel of probes such as single chromosome paints during a single operation. It should be useful for both chromosomal mapping projects and screening for chromosome abnormalities in clinical diagnostic laboratories.

Chromosomes↗
PubMed1994

YACs, BACs, PACs and MACs: artificial chromosomes as research tools.

Yeast artificial chromosomes (YACs) have become essential research tools as they enable large fragments of DNA to be cloned. In order to overcome several disadvantages of YACs, including chimaerism and instability, several complementary bacterial artificial chromosome (BAC) vectors have been developed. More recently, attempts are being made to construct artificial chromosomes in mammalian cells (MACs).

Animals↗
PubMed1994

De novo formation of several features of a centromere following introduction of a Y alphoid YAC into mammalian cells.

The DNA sequence requirements for mammalian centromere function have been investigated by re-introducing human YAC clones containing either centromeric or non-centromeric sequences into hamster and human cells. All YACs integrated into the host chromosomes. In most cell lines produced by spheroplast fusion into hamster cells, intact copies of the YAC and a large amount of yeast DNA were found. Cell lines produced by lipofection into human cells usually contained simple structures without yeast DNA. YACs containing Y alphoid DNA reformed several of the properties of a centromere, including a cytogenetically visible constriction, CREST antiserum binding and disruption of anaphase chromosome movement. In contrast, YACs containing non-centromeric sequences produced none of these results. This work suggests that a few hundred kb of alphoid DNA is sufficient to reconstitute several important features of a centromere.

Anaphase↗
PubMed1994

Addition of functional human telomeres to YACs.

Linear mammalian artificial chromosomes (MACs) will require functional telomeres, a centromere and the ability to replicate autonomously. We are investigating the possibility of developing MACs from yeast artificial chromosomes (YACs). Retrofitting vectors have been constructed to replace YAC telomeres with cloned human telomeric DNA. A modified YAC was introduced into mammalian cells by spheroplast fusion and the frequency with which the retrofitted human telomeric DNA seeded the formation of a new telomere was determined by Bal31 digestion and cytogenetic analysis. The telomere adjacent to the selectable marker gene was functional in 5/46 clones (11%) while the telomere 200 kb away at the other end of the YAC was functional in 1/46 clones (2%). These results indicate that despite the in vivo modification of the end of the telomere by the addition of yeast sequences, human telomeres will function at a high enough frequency to allow the construction of MACs by this route.

Chromosome Mapping↗
PubMed1993

A method for the generation of YAC transgenic mice by pronuclear microinjection.

Yeast artificial chromosomes (YACs) represent the latest generation of vectors which have the great advantage of large insert size. The introduction of YACs into mammalian cells and organisms has become an important goal, since it offers the potential to study the control of large and complex transcription units and identify genes by complementation. Microinjection into the nucleus is the most direct and efficient way of delivering YAC DNA into cells, but requires the purification of the YAC from the remaining yeast chromosomes. Here we describe a detailed method for the isolation of pure, intact and highly concentrated YAC DNA. As a model system the murine tyrosinase gene was chosen and four YACs covering this locus were isolated. Introduction by homologous recombination in yeast of sequences permitting YAC amplification greatly facilitated the isolation of YAC DNA at high concentrations. YAC DNA stabilized in a salt and polyamine containing buffer did not compromise the survival of microinjected oocytes and was suitable for the generation of transgenic mice. Applications and benefits of this technique will be discussed.

Animals↗
PubMed1993

Physical mapping and YAC contig analysis of the region surrounding Xist on the mouse X chromosome.

The Xist sequence has been proposed as a potential candidate for the X-inactivation center based both on its localization within the candidate region for the X-inactivation center in man and mouse and on its unique pattern of expression from the inactive X chromosome. We have cloned 550 kb of DNA surrounding the mouse Xist sequence in contiguously overlapping YAC clones and have developed a long-range restriction map that spans almost 1 Mb of this region and includes this YAC contig. The detailed restriction map we have established provides a framework for the identification of expressed sequences other than Xist that may equally exhibit unusual expression characteristics associated with X inactivation. The presence of possible structural or methylation differences within this region between the active and inactive X chromosomes has been investigated through comparative analysis of male and female genomic DNA, and we report here the identification of certain CpG-containing restriction sites around Xist that have an interesting differential methylation status on the inactive and active X chromosomes.

Animals↗