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Biomedical subjects

A J Richards

Publications and source records attributed to A J Richards.

At least 19 recordsLinked to original sources
PubMed1992

A single base mutation in the gene for type III collagen (COL3A1) converts glycine 847 to glutamic acid in a family with Ehlers-Danlos syndrome type IV. An unaffected family member is mosaic for the mutation.

Ehlers-Danlos syndrome type IV, an inherited connective tissue disease, is usually caused by mutations in the gene for type III collagen. Here, we describe a glycine to glutamic acid substitution in a patient with this syndrome. Previous studies had shown that fibroblasts from the patient, his mother and brother secreted a reduced amount of type III collagen and also produced an overmodified form of the protein that was preferentially retained intracellularly. Peptide mapping experiments indicated that the mutation was located within cyanogen bromide peptide 9. This was supported by chemical cleavage analysis and sequencing of cDNA encoding this region. Allele-specific oligonucleotide hybridisation of genomic DNA confirmed that a G to A mutation converted Gly 847 to Glu. The mutation was present in two other affected family members and also in a third, who was clinically unaffected. Further analysis of this unaffected individual revealed reduced mutant:normal ratios in DNA obtained from both blood and hair samples, showing that she was mosaic for the mutation.

Amino Acid Sequence↗
PubMed1992

A 27-bp deletion from one allele of the type III collagen gene (COL3A1) in a large family with Ehlers-Danlos syndrome type IV.

A large family with Ehlers-Danlos syndrome type IV (EDS IV) has previously been described. Unlike most cases of EDS IV, fibroblasts from affected members secreted near normal amounts of type III collagen. We have localized the mutation in this family to the CB5 peptide of type III collagen, by using both protein and cDNA mapping techniques. Sequence analysis of cDNA revealed a 27-bp deletion within exon 37, a deletion that removed nine amino acids and maintained the Gly-X-Y repeat of the collagen helix. Further sequencing of genomic DNA confirmed its location, and amplification of DNA from family members showed that it was absent in unaffected individuals but present in all the affected individuals tested. This deletion is flanked by two short direct repeats of CTCC; it may have arisen by slipped mispairing, and has subsequently been transmitted to all affected family members.

Alleles↗
PubMed1991

Characterisation of a glycine to valine substitution at amino acid position 910 of the triple helical region of type III collagen in a patient with Ehlers-Danlos syndrome type IV.

We have studied a patient with Ehlers-Danlos syndrome type IV. Protein mapping studies of her type III collagen had indicated that cyanogen bromide fragment 9 contained the site of the mutation. Here we describe the mapping of this region for a single base mutation using a chemical modification and cleavage technique. Sequence analysis of cDNA showed a G to T mutation resulting in the substitution of glycine 910 by valine. This was confirmed by allele specific oligonucleotide hybridisation to the proband's genomic DNA.

Alleles↗
PubMed1990

Developmental regulation of cell-type-enriched mRNAs in Dictyostelium discoideum.

We describe sixteen new families of cDNA clones representing mRNAs that are expressed preferentially in either prespore or prestalk cells during development of Dictyostelium discoideum and two new mRNAs that are expressed in a non-cell-type-specific manner. None of the prespore-enriched mRNAs are detectable in Dictyostelium cells until 13-15 h of development but then they increase dramatically and peak at 18-22 h. Upon dissociation of developing aggregates, all these mRNAs rapidly decay to low levels. In marked contrast to data presented for prespore genes by other workers, cyclic AMP either has no effect on the mRNA levels in dissociated cells or is only weakly effective in restoring normal expression. A prestalk-enriched mRNA examined, 5G mRNA, is similarly expressed late in development but is also expressed in vegetative cells. The level of 5G mRNA is only moderately affected by cell disaggregation.

Cyclic AMP↗
PubMed1990

Developmental expression and characterization of the gene encoding spore coat protein SP60 in Dictyostelium discoideum.

The complete coding sequence, upstream sequence and developmental expression of Dictyostelium discoideum AX2 spore coat protein gene SP60 is reported. The gene contains two exons, 154bp and 1121bp long, separated by a 119bp intron, and encodes a protein of 46,925 molecular weight plus a 23-amino-acid hydrophobic leader sequence. The N-terminus of the mature protein consists of four copies of a perfect hexapeptide repeat (GDWNNN). The central region is rich in cysteine residues, including four highly conserved cysteine-rich repeats with homology to 'EGF-like' repeats. The C-terminus is aspartate-rich and composed of multiple imperfect copies of a D(G/D)DYD repeat followed by several repeats of the tetrapeptide DNDW and derived sequences. A TATA box promoter motif juxtaposed to an oligo(dA) stretch lies 52bp upstream of the main transcriptional start site of the gene. Six AC-rich boxes occur in the region -327 to -556, all of which contain the consensus sequence CACAC. Two GC-rich boxes and a C-rich element (TTACCCCA) are also present upstream. Another open reading frame is positioned a short distance downstream of the SP60 gene in the opposite transcriptional orientation. Expression of the SP60 gene ceases upon disaggregation to single cells and cannot be restored by high levels of extracellular cAMP either alone or in combination with conditioned medium factors.

Amino Acid Sequence↗
PubMed1990

Cell-type-specific genes expressed late in Dictyostelium development show markedly different responses to 3'5' cyclic AMP.

The maintenance of late gene expression by 3'5' cyclic AMP was re-examined using several newly isolated cell-type-specific genes. Expression of all the prespore-enriched genes ceased immediately upon disaggregation of developing cells and pre-existing mRNA was rapidly degraded. For most genes, cAMP had little or no effect either alone or in combination with conditioned medium factors. The expression of the non-cell-type-specific genes 7E and 2C also ceased upon cell disaggregation but cAMP triggered a full re-induction of expression although the timing of the response differed markedly between these two genes. In contrast to earlier interpretations, these data argue that for none of these late prespore genes is cAMP alone sufficient for the maintenance of expression. The responses of the two prestalk mRNAs examined were gene-specific. Prestalk 5D mRNA decayed slowly upon disaggregation and was partially stabilized by cAMP whereas prestalk 5G mRNA increased upon disaggregation and was inhibited by cAMP.

Culture Media↗
PubMed1990

Two cyclic AMP-regulated genes from Dictyostelium discoideum encode homologous proteins.

Expression of the 7E and 2C genes late in Dictyostelium development ceases upon cell disaggregation but, in contrast to many other genes we have studied, expression is fully restored by exogenous cAMP (A. J. Richards et al., submitted). The 7E and 2C genes encode polypeptides of similar size (9220 and 10573 Daltons, respectively), each of which contains an unusually high proportion of serine plus glycine residues (41% and 59%, respectively). Each protein possesses a relatively serine-rich N-terminus and glycine-rich C-terminus and contains the conserved sequence S(X)SSS(X2)SS(X)SS(X2)SFGS. These data suggest that genes 7E and 2C may have arisen by duplication of a common ancestor. Computer analysis indicates that both gene products are probably intracellular structural proteins that form extended coil structures.

Amino Acid Sequence↗
PubMed1985

A study of one of the iron-sulphur clusters in oxidized hydrogenase from Megasphaera elsdenii by magnetic-circular-dichroism spectroscopy.

The m.c.d. spectrum of the oxidized state of hydrogenase from Megasphaera elsdenii has been measured at liquid-helium temperatures. This oxidation state of the enzyme displays a characteristic rhombic e.p.r. signal with g-values of 2.101, 2.052 and 2.005 assigned previously to a [4Fe-4S]3+ cluster as in oxidized HiPIP (high-potential iron-sulphur protein) [Van Dijk, Grande, Mayhew & Veeger (1980) Eur. J. Biochem. 107, 251-261]. The low-temperature m.c.d. spectrum shows no features attributable to an oxidized four-iron cluster of the HiPIP type, but does reveal broad, positive peaks at 460 and 730 nm, which magnetize in a manner untypical of a spin S = 1/2 cluster with g-values close to 2. The m.c.d. spectrum is most closely similar to that of dye-oxidized P-clusters known in the enzyme nitrogenase. It is therefore proposed that the rhombic e.p.r. spectrum at a g-value close to 2 arises from an m.c.d.-silent radical species that may be related chemically to the cysteine persulphide species, RS-S., recently found in the hexacyanoferrate-oxidized seven-iron ferredoxin of Azotobacter vinelandii [Morgan, Stephens, Devlin, Stout, Melis & Burgess (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1931-1935].

Circular Dichroism↗