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Biomedical subjects

N Affara

Publications and source records attributed to N Affara.

At least 19 recordsLinked to original sources
PubMed1989

Molecular heterogeneity of steroid sulfatase deficiency: a multicenter study on 57 unrelated patients, at DNA and protein levels.

Steroid sulfatase (STS) deficiency is the biochemical defect of X-linked ichthyosis (XLI), one of the most common X-linked disorders. We studied 57 European unrelated patients affected by STS deficiency. Twenty-eight patients were from Italy, 24 from the United Kingdom, 4 from The Netherlands, and 1 from Denmark. In two families XLI was associated with Kallmann syndrome (hypogonadotropic hypogonadism and anosmia). STS enzymatic activity was profoundly deficient in all cases. Direct DNA analysis, using cDNA and genomic probes from the STS gene and linked regions, demonstrated heterogeneity of the molecular defect. Forty-eight patients (84%) showed a deletion of the STS gene. In 44 cases the deletion also involved the STS flanking locus DXS237. In 1 patient a partial deletion of the STS gene was detected and in 9 patients no evidence of deletion was found. Locus DXS31 (probe M1A), previously mapped to Xp22.3-pter, was not deleted either in 24 patients with X-linked ichthyosis or in two families with X-linked ichthyosis associated with Kallmann syndrome. Consequently, the following loci order could be suggested: telomere--DXS31--(DXS237, STS)--Kallmann--centromere. Immunoblotting experiments, performed using anti-STS polyclonal antibodies, revealed the absence of cross-reacting material to STS in all cases tested, including 4 patients without evidence of deletions.

Arylsulfatases↗
PubMed1989

Localization of murine X and autosomal sequences homologous to the human Y located testis-determining region.

Recently a candidate gene for the primary testis-determining factor (TDF) encoding a zinc finger protein (ZFY) has been cloned from the human Y chromosome. A highly homologous X-linked copy has also been identified. Using this human sequence it is possible to identify two Y loci, an X and an autosomal locus in the mouse (Zfy-1, Zfy-2, Zfx and Zfa, respectively). Suprisingly ZFY is more homologous to the mouse X and autosomal sequences than it is to either of the Y-linked loci. Both Zfy-1 and Zfy-2 are present in the Sxr region of the Y but Zfy-2 is absent in the Sxr deletion variant Sxrb (or Sxr") suggesting it is not necessary for male determination. Extensive backcross analyses map Zfa to mouse chromosome 10 and Zfx to a 5-cM interval between anonymous X probe MDXS120 and the tabby locus (Ta). We also show that the mouse androgen receptor locus (m-AR) believed to underlie the testicular feminization mutation (Tfm) shows complete linkage to Zfx. Comparative mapping indicates that in man these genes lie in separate conserved DNA segments.

Animals↗
PubMed1989

Molecular characterization of human X/Y translocations suggests their aetiology through aberrant exchange between homologous sequences on Xp and Yq.

Several DNA sequences from two homologous regions, localized on the distal part of the human X chromosome short arm and on the long arm of the Y chromosome, have been hybridized to DNAs from seven human-rodent hybrids containing human X; Y translocation chromosomes. Molecular characterization of the translocated chromosomes has revealed, in all but one case, transfer of the Y cluster of sequences and complete deletion of the corresponding X-chromosomal sequences. The possible role of X/Y homology in the aetiology of X; Y translocations is proposed.

Cell Line↗
PubMed1988

Expression of chromosome 21 specific sequences in normal and Down's syndrome tissues.

Using RNA isolated from age and sex matched normal and Down's Syndrome foetal liver and brain tissues, Northern blots were prepared and probed with 4 chromosome 21 specific sequences. The results show that no consistent pattern of expression emerges when Down's Syndrome tissue is compared with normal tissue but the results are very different from the 3/2 ratio of expression which may be expected. Two sequences 21.3 and 26C show only minor differences in expression in trisomy 21 liver samples but significant changes in their expression pattern when normal and Down's Syndrome brain samples are compared. The other sequences, JG77 and JG90 show a 5 fold higher degree of expression in Down's brain but when liver samples are compared one of these sequences shows equal levels of expression in normal and Down's Syndrome samples and the other shows a decrease in expression level in Down's Syndrome samples.

Animals↗
PubMed1988

Mapping of 12 translocation breakpoints in the Xp21 region with respect to the locus for Duchenne muscular dystrophy.

Over 20 females have been reported to carry reciprocal X; autosome translocations with breakpoints in Xp21 and to suffer from Duchenne muscular dystrophy (DMD). We have positioned nine of these breakpoints with respect to the Duchenne gene by mapping probes from the DMD region against a panel of somatic cell hybrids, each containing one of the translocation chromosomes from a different female patient; further information has also been obtained by in situ hybridization, including the breakpoint location in a tenth DMD patient. We have also characterized two translocation breakpoints that lie in the same chromosomal region but which are not associated with the expression of DMD. All the DMD-associated translocation breakpoints examined lie at several sites within the DMD locus and between the two non-DMD breakpoints.

Cell Line↗
PubMed1986

The structure of the mouse glutathione peroxidase gene: the selenocysteine in the active site is encoded by the 'termination' codon, TGA.

Glutathione peroxidase (GSHPx) is an important selenium-containing enzyme which protects cells from peroxide damage and also has a role in leukotriene formation. We report the identification of a genomic recombinant as encoding the entire mouse GSHPx gene. Surprisingly, the selenocysteine in the active site of the enzyme is encoded by TGA: this has been confirmed by primer extension/dideoxy sequencing experiments using reticulocyte mRNA. The same site of transcription initiation is used in three tissues in which the GSHPx mRNA is expressed at high levels (erythroblast, liver and kidney). Like some other regulated 'house-keeping' genes, the GSHPx gene has Sp1 binding site consensus sequences but no 'ATA' and 'CAAT' consensus sequences upstream of the transcription initiation site. Moreover, there is a cluster of two Sp1 binding site consensus sequences and two SV40 core enhancer sequences in the 3' region of the gene, close to the previously mapped position of a DNase I-hypersensitive site found only in tissues expressing the GSHPx mRNA at high levels.

Amino Acid Sequence↗
PubMed1985

Analysis of chromatin changes associated with the expression of globin and non-globin genes in cell hybrids between erythroid and other cells.

Red blood cell differentiation involves the coordinate expression of a set of polypeptides some of which are erythroid-specific (the abundant globins as well as minor species such as glycophorin, carbonic anhydrase I and the RBC lipoxygenase) whereas others are found also in a subset of other cells, e.g. beta spectrin and a 19 kd polypeptide (ep 19) found in adult liver and kidney as well as erythroid cells. To investigate the genetic mechanisms involved in the regulation of these classes of genes, the expression of lipoxygenase, ep 19 and beta globin mRNAs was investigated in cell hybrids between mouse erythroid (Friend) cells and mouse T-lymphoma or neuroblastoma cells. All three mRNAs are expressed or repressed together in cell hybrids between the Friend cell and lymphoma or neuroblastoma cells respectively. Moreover, studies of the chromatin structure surrounding the genes reveal that erythroid cell-specific DNaseI hypersensitive sites within the ep 19 and beta major globin genes are lost in the Friend cell X neuroblastoma hybrids whereas they are retained in the Friend cell X lymphoma cell hybrids. This implies that the trans-acting mechanism responsible for regulating the RBC phenotype in these cell hybrids acts at the level of the early chromatin changes thought to reflect a pre-activation stage in gene expression.

Animals↗
PubMed1983

Isolation of non-globin genes expressed preferentially in mouse erythroid cells.

Genomic DNA recombinants were isolated from a library of Balb-C mouse genomic DNA fragments cloned in lambda Ch4A by screening with cDNA derived from 13d foetal liver cell or adult reticulocyte poly A+ RNA. Subsequent screening enabled us to identify non-globin genomic sequences whose expression appeared exclusive to or elevated in erythroid cells. Further analysis of the structure and expression of these sequences was performed using Southern blot and DNA or RNA dot hybridisation analysis. In one recombinant part of the cloned genomic sequence corresponded to an erythroblast specific mRNA identified previously by Affara et al, (5).

Animals↗
PubMed1983

Patterns of expression of erythroblast non-globin mRNAs.

Three classes of erythroblast non-globin mRNAs have been identified using cDNA recombinants isolated from a mouse foetal liver cDNA library. One erythroid cell-specific 12S mRNA coding for a 16-19,000 dalton protein has been identified using two independent cDNA recombinants (pFC5 and pFA6). The gene encoding this mRNA is unique in the genome and its organisation seems to be the same in both erythroid and non erythroid cell genomic DNA as judged by digestion with restriction enzymes and Southern blotting. Another erythroblast mRNA of size 16S represented by recombinant pFD12 is expressed in brain cells as well as a variety of haemopoietic cell types, but not in adult liver or fibroblasts. Two other erythroblast mRNAs of size 8S and 12S represented by recombinants pD6 and pA4 are expressed in many differentiated cell types with the exception of non-dividing cells such as reticulocytes, peripheral white blood cells, adult liver and brain cells. These mRNAs are, therefore, presumed to be involved ubiquitously in cell proliferation or general cell metabolism.

Animals↗
PubMed1979

Biochemical aspects of muscle differentiation as analyzed by in vitro cultivation techniques.

Myoblasts cultivated in vitro will undergo terminal differentiation to form muscle fibres. Teratoma derived mouse cell lines, a pluripotent primitive line, and a muscle cell line, provide a possibility for comparing RNA populations in an early precursor cell with those in committed myoblasts and differentiated myotubes. Molecular hybridization analyses led to the conclusion that new RNA sequences appear in the cytoplasm during muscle differentiation. Such muscle specific sequences are not detectable in the nuclear RNA of myoblasts or primitive cells. Studies of protein synthesis during terminal myogenesis indicate co-ordinate expression of the muscle contractile proteins. These represent distinct isozymes, distinguishable from the contractile proteins of other cell types. In the case of myosin light chains isozymic transitions between different muscle forms have been identified during early development.

Animals↗