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D L Kaufman

Publications and source records attributed to D L Kaufman.

44 records · Page 3Linked to original sources
PubMed1990

Splicing defect at the ornithine aminotransferase (OAT) locus in gyrate atrophy.

Gyrate atrophy (GA), a recessive eye disease involving progressive vision loss due to chorioretinal degeneration, is associated with the deficiency of the mitochondrial enzyme ornithine aminotransferase (OAT), with consequent hyperornithinemia. We and others have reported a number of missense mutations at the OAT locus which result in GA. Here we report a GA patient of Danish/Swedish ancestry in whom one OAT allele produces an mRNA that is missing a single 96-bp exon relative to the normal mRNA. Polymerase-chain-reaction amplification and sequencing revealed a 9-bp deletion covering the splice acceptor region of exon 5, resulting in the absence of exon 5 sequences from the mRNA with no disruption to the reading frame. This mutation, which was not present in 15 other independent GA patients, adds to the array of allelic heterogeneity observed in GA and represents the first example of a splicing mutation associated with this disorder.

Adult↗
PubMed1987

Glutamic acid decarboxylase cDNA: nucleotide sequence encoding an enzymatically active fusion protein.

Glutamic acid decarboxylase (GAD;E.C. 4.1.1.15) catalyzes the production of GABA, the major inhibitory neurotransmitter in the mammalian brain. We recently isolated a lambda gt-11 recombinant, lambda-GAD, that contains the cDNA for GAD from feline brain (Kaufman et al., 1986). Interestingly, the beta-galactosidase-GAD fusion protein encoded by lambda GAD is enzymatically active, catalyzing the conversion of glutamate to CO2 and GABA. Here we report the nucleotide sequence of feline GAD cDNA. It consists of 2265 bases, with a continuous open reading frame of 625 codons. The derived sequence contains the sequence Asn-Pro-His-Lys, which is identical to sequence at the pyridoxal phosphate-binding site of porcine DOPA decarboxylase (Bossa et al., 1977). The first ATG sequence in the open reading frame begins at nucleotide residue 118. The 585 codons 3' to this putative initiation site predict an amino acid composition, N-terminal residue, and molecular size consistent with published characterizations of GAD.

Amino Acid Sequence↗
PubMed1986

Brain glutamate decarboxylase cloned in lambda gt-11: fusion protein produces gamma-aminobutyric acid.

Glutamate decarboxylase (GAD; E.C. 4.1.1.15) converts glutamate to gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the vertebrate central nervous system. This report describes the isolation of a GAD complementary DNA clone by immunological screening of a lambda gt-11 brain complementary DNA expression library. The fusion protein produced by this clone catalyzes the conversion of glutamate to GABA and carbon dioxide, confirming its identity as GAD. Antibodies to beta-galactosidase remove GAD enzymatic activity from solution, showing that this activity is associated with the fusion protein. In immunoblotting experiments all three available antisera to GAD reacted with the fusion polypeptide and with two major polypeptides (molecular size, 60,000 and 66,000 daltons) in brain extracts.

Animals↗
PubMed1986

In situ hybridization to localize mRNA encoding the neurotransmitter synthetic enzyme glutamate decarboxylase in mouse cerebellum.

Glutamate decarboxylase (GAD; EC 4.1.1.15) is responsible for the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA). We have used a cDNA sequence encoding GAD to produce a single-stranded RNA hybridization probe for GAD mRNA. This probe detects GAD mRNA in individual cells in sections of mouse cerebellum. The specificity of in situ hybridization with this probe rests on four criteria: the distribution of labeled cells matched the results we and others obtain with GAD immunohistochemistry (Purkinje, Golgi II, stellate, and basket neurons were labeled, whereas granule cells and glia were not); a negative control probe having a sequence identical to GAD mRNA did not specifically label any cerebellar cells; prior treatment of the sections with RNase abolished specific labeling; the labeling showed the melting behavior typical of nucleic acid hybrids. Translation of GAD mRNA is apparently restricted to neuronal cell bodies since GAD mRNA was detectable in neuronal perikarya but not in terminals. Also, the choice of GABA as a neurotransmitter appears to be made at the level of transcription since granule neurons did not contain detectable GAD mRNA. The level of GAD mRNA varied among the classes of neurons as well as from cell to cell within each neuron type.

Animals↗
PubMedDate not supplied

Horizontal cells in cat and monkey retina express different isoforms of glutamic acid decarboxylase.

The neurotransmitter used by horizontal cells in mammals has not been identified. GABA has been the leading candidate, but doubt has remained because of failure to clearly demonstrate the GABA synthetic enzyme, glutamic acid decarboxylase (GAD) in these cells. Because GAD was recently shown to exist as two isoforms, 65 kDa and 67 kDa, we considered whether there might be a mismatch between the forms of GAD expressed in horizontal cells and the probes used to detect it. Accordingly, we stained sections of mammalian retina with antibodies specific for each isoform. Cat horizontal cells of both types (A and B) were immunoreactive for GAD67 but negative for GAD65; monkey horizontal cells of both types (H(I) and HII) were positive for GAD65 and negative for GAD67. The findings reconcile previous, apparently conflicting, observations and strengthen considerably the hypothesis that mammalian horizontal cells are GABAergic.

Animals↗