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J E Mejía

Publications and source records attributed to J E Mejía.

8 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↗
PubMed1997

Retrofitting vectors for Escherichia coli-based artificial chromosomes (PACs and BACs) with markers for transfection studies.

P1-based artificial chromosomes (PACs) and bacterial artificial chromosomes (BACs) have significantly expanded the size of fragments from eukaryotic genomes that can be stably cloned in Escherichia coli as plasmid molecules. Functional characterization of a gene within a given PAC or BAC clone often requires transferring the DNA into eukaryotic cells for transient or long-term expression. To facilitate transfection studies, we have developed protocols using the Notl restriction sites of any PAC or BAC clone to introduce a transfection reporter gene, lacZ, together with a selectable marker, neo. This enables transfected cells to be detected by X-Gal staining to verify DNA uptake, and clones of stably transformed cells may be selected for in the presence of the antibiotic G418. The same retrofitting protocols may be applied with other markers of interest to extend the functionality of PAC and BAC libraries, and specialized aspects of such manipulation of E. coli-based artificial chromosomes are outlined.

Bacteriophage P1↗
PubMed1995

DNA methylation and the origin of complement factor B polymorphism.

BF is a polymorphic complement component encoded in the MHC. In each of two frequent alleles of BF, BF*FA and BF*FB, the difference in relation to the major allele BF*S has been shown to consist in the nonsynonymous substitution of only one base of the coding sequence. Both substitutions occur within the same codon and affect contiguous positions, corresponding to the dinucleotide CpG in BF*S. We propose here that BF*FA and BF*FB arose independently from BF*S by the frequently described transition mutations associated with cytosine methylation at CpG sites. By probing sperm DNA with methylation-sensitive restriction enzymes, we obtained experimental evidence of germ line methylation of the CpG site considered. The dinucleotide of the BF gene probably constitutes a site for recurrent mutation, and this is of relevance for the use of BF as a genetic marker, and the origin of forms of the protein with altered functional properties.

Base Sequence↗
PubMed1994

Human factor B. Complete cDNA sequence of the BF*S allele.

The gene of human complement factor B (BF) is located within the class III region of the major histocompatibility complex. The knowledge of the coding sequence of the BF gene rests on a set of partial sequence studies reported by various sources, and full-length sequences ascribed to specific alleles of this polymorphic complement component have not yet been published. Now, we have isolated and sequenced a collection of cDNA clones derived from BF*S, the major BF allele. We present an uninterrupted, allele-specific sequence of the entire coding region and the 3' untranslated segment of the cDNA. Extensive comparison of this and previously available sequence data was carried out, and a number of base substitutions were observed in relation to some of the earlier sequences. The possibility that these differences arise from polymorphism in the BF gene is discussed.

Alleles↗
PubMed1994

Genomic analysis of the F subtypes of human complement factor B.

Factor B of human complement is encoded within the Major Histocompatibility Complex (MHC) and is polymorphic, with up to 30 alleles defined by electrophoretic mobility. One of the most common alleles, BF*F, is subdivided into the FA and FB subtypes, which differ at the gene level by non-synonymous base substitutions in the seventh codon. We have found at this position a new restriction site polymorphism, as a Bsl I site absent from the FB allele. Using this restriction polymorphism, we have developed a method for BF F subtype determination, based on amplification by polymerase chain reaction of the 5' end of the BF gene, and digestion with Bsl I. This new method has been applied to a panel of 29 selected BF F individuals. A single strand DNA conformation analysis of the same region of the gene allowed us to confirm the above DNA-based BF F subtyping. During this study, two BF*F1 alleles showed discrepancies between protein and DNA typing, which were confirmed by our sequencing data. These were identical, in the 5' region, to BF*S and BF*FB genes, respectively. In a comparison with two protein subtyping methods, identical results were found for only one third of the selected samples. The conflicting results may arise, in part, from previously undescribed molecular heterogeneity within BF F subtypes, or from the presence of a null allele. Our new method allows BF*F subtyping to be used with confidence in the definition of disease-associated MHC haplotypes.

Alleles↗