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Biomedical subjects

M E Case

Publications and source records attributed to M E Case.

At least 55 records · Page 3Linked to original sources
PubMed1975

Genetic evidence on the organization and action of the qa-1 gene product: a protein regulating the induction of three enzymes in quinate catabolism in Neurospora crassa.

The first three reactions in the catabolism of qainic acid in Neurospora crassa are under the genetic control of the qa gene cluster. This cluster consists of three structural genes encoding three inducible enzymes plus a regulatory gene (qa-1+) whose diffusible product apparently acts in a positive fashion to initiate coordinate synthesis of the three enzymes when an appropriate inducer is present. Genetic and biochemical evidence for both complementing and temperature-sensitive qa-1 alleles indicates that the product of the qa-1+ gene is an oligomeric (multimeric) protein. On the basis of cis-trans tests of appropriate double mutants (plus genetic mapping data for temperature-sensitive mutants), at least certain constitutive mutants (which produce all three qa enzymes in the absence of an inducer) are mutants in the regulatory gene and not in controlling elements such as initiators. The detection of stable (non-revertible) qa-1 intralocus deletion (multisite)mutants provides additional evidence for positive regulation in the qa system. Extensive genetic recombination data provide evidence that the two types of qa-1 mutants--slow-complementing (qa-1-s) and fast-complementing (qa-1-f)--map in discrete, non-overlapping segments of the qa-1 locus. These two distinct types of mutants are hypothesized to produce altered regulatory protein molecules that fail to interact either with a DNA initiator site (qa-1-s types) or with an inducer (qa-1-f types). The striking similarities between the qa system in this lower eukaryote and certain prokaryote operon systems are discussed.

Alcohol Oxidoreductases↗
PubMed1975

Effect of mutations in the qa gene cluster of Neurospora crassa on the enzyme catabolic dehydroquinase.

Catabolic dehydroquinase, which functions in the inducible quinic acid catabolic pathway of Neurospora crassa, has been purified from wild type (74-A) and three mutants in the qa gene cluster. The mutant strains were: 105c, a temperature-sensitive constitutive mutant in the qa-1 regulatory locus; M-16, a qa-3 mutant deficient in quinate dehydrogenase activity; and 237, a leaky qa-2 mutant which possess very low levels of catabolic dehydroquinase activity. The enzymes purified from strains 74-A, 105c, and M-16 are identical with respect to behavior during purification, specific activity, electrophoretic behavior, stability, molecular weight, subunit structure, immunological cross-reactivity, and amino acid content. The mutant enzyme from strain 237 is 1,500-fold less active and appears to have a slightly different amino acid content. It is identical by a number of the other criteria listed above and is presumed to be a mutant at or near the enzyme active site. These data demonstrate that the qa-1 gene product is not involved in the posttranslational expression of enzyme activity. The biochemical identity of catabolic dehydroquinase isolated from strains 105c and M-16 with that from wild type also demonstrates that neither the inducer, quinic acid, nor other enzymes encoded in the qa gene cluster are necessary for the expression of activity. Therefore the combined genetic and biochemical data on the qa system continue to support the hypothesis that the qa-1 regulatory protein acts as a positive initiator of qa enzyme synthesis.

Alcohol Oxidoreductases↗
PubMed1974

Temperature-sensitive pleiotropic revertants from a mutant in the arom gene cluster of Neurospora crassa.

Genetical and biochemical studies have been performed with revertants induced in a polyaromatic mutant (No. 58) in the arom gene cluster of Neurospora crassa. In addition to complete and partial revertants able to grow on minimal at both 25 degrees and 35 degrees , temperature-sensitive revertants capable of growth on minimal at 25 degrees but not at 35 degrees have been recovered. One of these revertants has been shown to lack biosynthetic dehydroquinase activity at both temperatures (utilizing the inducible catabolic isozyme for growth at 25 degrees ), to have dehydroshikimate reductase activity only at 25 degrees , and to form an arom aggregate having a molecular weight approximately one-half that of wild type. These results are interpreted as indicating that pleiotropic mutants in the arom gene cluster can result from missense mutations, as well as from nonsense mutations as indicated in previous studies.

Alcohol Oxidoreductases↗
PubMed1974

Revertants and secondary arom-2 mutants induced in non-complementing mutants in the arom gene cluster of Neurospora crassa.

Extensive genetical and biochemical studies have been performed with revertants and secondary arom-2 mutants induced in two different primary non-complementing mutants which map within the arom gene cluster of Neurospora crassa. These studies indicate that mutant M54 but not M25 can revert by super-suppressor mutations in unlinked genes, thus confirming previous evidence that M54 contains a nonsense codon. At least three new super suppressors of M54 have been detected. All four super suppressors (including one previously detected) when combined with M54 result in high levels of all five of the arom enzymic activities in the form of arom multienzyme complexes very similar to (but not necessarily identical with) that in wild type (WT).-Evidence has also been obtained that the two non-complementing mutants can yield revertants which appear to result from true back mutations and produce arom aggregates essentially indistinguishable from that of WT. In addition, M25, but not M54, when plated on quinic acid yields revertants (secondary mutants) some of which are phenotypically indistinguishable from arom-2 primary mutants and others of which, although also mapping within the arom-2 gene, exhibit unusual properties. Genetic evidence indicates that the M25 secondary mutants are localized within the arom-2 gene, but that they arise from mutational events more complex than ones resulting in single base pair changes in the M25 codon.-The recovery of secondary arom-2 mutants as revertants of non-complementing arom mutants provides strong evidence, independent of earlier recombination data, that non-complementing arom mutants are located within the arom-2 structural gene of the arom gene cluster. In addition, the occurrence and characteristics of these secondary arom-2 mutants provide strong evidence, independent of the results with nonsense suppressors, that the arom gene cluster is transcribed, beginning with the arom-2 gene, as a single polycistronic messenger ribonucleic acid (mRNA) molecule which is subsequently translated into the arom multienzyme complex.

Alcohol Oxidoreductases↗
PubMed1972

Direct induction in wild-type Neurospora crassa of mutants (qa-1 c ) constitutive for the catabolism of quinate and shikimate.

A color test has been developed for the selection and identification of mutants in Neurospora crassa, constitutive for the three normally inducible enzymes which convert quinate to protocatechuate. By this means seven such mutants have been recovered after ultra violet irradiation of wild type and have been shown to be allelic (or very closely linked) to the qa-1(C) mutants previously obtained by other means. Thus, the regulation of the synthesis of these three catabolic enzymes is indicated to be under the control of a single gene, qa-1(+).

Alcohol Oxidoreductases↗
PubMed1971

Partial enzyme aggregates formed by pleiotropic mutants in the arom gene cluster of Neurospora crassa.

Molecular weights of enzymically active arom aggregates produced by pleiotropic and various arom-1 mutants of Neurospora crassa have been estimated by sucrose density gradient centrifugation. In contrast to most single-gene mutants (which produce intact arom multienzyme aggregates of normal molecular weight- about 230,000), pleiotropic mutants that lack two or more of the five enzyme activities in the arom aggregate produce partial arom aggregates of molecular weights ranging from about 60,000 to about 85,000. In addition, certain arom-1 mutants are pleiotropic in producing arom aggregates of about half the normal molecular weight. Experiments with heterocaryons, as well as with mixtures of extracts from various mutants, have provided evidence concerning the presence or absence of interactions (as detectable molecular hybridization) between different arom aggregates. An evaluation has been made of the relation between the genetic location of particular mutants within the arom gene cluster and the size and enzymic content of the arom aggregates they produce. Interpretations concerning the molecular organization of the arom multienzyme aggregate in N. crassa are presented.

Centrifugation, Density Gradient↗
PubMed1971

Constitutive mutants in a regulatory gene exerting positive control of quinic acid catabolism in Neurospora crassa.

In Neurospora crassa, evidence has recently been obtained for a cluster of four closely-linked genes controlling the inducible enzymes catalyzing the first three reactions in the catabolism of quinic acid. Three of these genes appear to be the structural genes for the three enzymes. The fourth gene, designated qa-1, has been interpreted as having a regulatory function, since qa-1 mutants are pleiotropic types, are noninducible for the three enzymes, and form heterocaryons which complement mutants in the structural genes. The present studies were undertaken to elucidate further the nature of the regulatory role of the qa-1 locus. A number of constitutive (qa-1(C)) mutants have been obtained from certain qa-1 mutants as revertants selected for their ability to grow on quinic acid as a sole source of carbon. These qa-1(C) mutants produce high levels of all three enzymes in the absence of an inducer, map within (or very close to) the qa-1 locus, and produce a catabolic dehydroquinase (EC 4.2.1.10) which is indistinguishable, on the basis of thermolability tests, from that of wild type. In addition, when grown in the absence of an inducer, heterocaryons between wild-type (qa-1(+)) and different qa-1(C) mutants exhibit markedly different levels of constitutivity (from 5 to 50% of the particular parental qa-1(C) mutant) for catabolic dehydroquinase, one of the enzymes under qa-1 control. These overall results are interpreted as supporting the hypothesis that the qa-1(+) gene product (presumably a multimeric protein) plays only a positive regulatory role in initiating synthesis of the three quinate catabolic enzymes.

Alcohol Oxidoreductases↗
PubMedDate not supplied

Cloning the quinic acid (aq) gene cluster from Neurospora crassa: identification of recombinant plasmids containing both qa-2+ and qa-3+.

A 22.2-kb insert of Neurospora crassa DNA containing at least two of the genes from the inducible catabolic quinic acid pathway has been cloned into the cosmid vehicle pHC79 resulting in a recombinant plasmid, pMSK308. The qa-2+ locus (which encodes catabolic dehydroquinase) is functionally expressed in both Escherichia coli and qa-2 mutants of N. crassa transformed with pMSK308 plasmid DNA. Expression of the qa-3 gene (which encodes quinate dehydrogenase) is only detected upon reintroduction into N. crassa. Results were also obtained which suggested that the qa-4 gene, which maps between qa-2 and qa-3, may also be present on both pMSK308 and the previously described plasmid pVK88. Certain anomalies in the types of N. crassa transformants obtained with pMSK308 plasmid DNA were noted.

Alcohol Oxidoreductases↗