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Y H Fu

Publications and source records attributed to Y H Fu.

60 records · Page 4Linked to original sources
PubMed1988

Metabolic control and autogenous regulation of nit-3, the nitrate reductase structural gene of Neurospora crassa.

In Neurospora crassa, the expression of nit-3, the structural gene which encodes nitrate reductase, is highly regulated and requires both nitrate induction and nitrogen catabolite derepression. The major nitrogen regulatory gene, nit-2, acts in a positive fashion to turn on the expression of nit-3 and other nitrogen-related genes during nitrogen derepression. A second regulatory gene, designated nmr, acts in a negative fashion to repress the expression of nitrate reductase and related enzymes, and nmr mutants are partially insensitive to nitrogen repression. Using cloned genes as specific hybridization probes, we demonstrated that nmr does not affect the transcription of nit-2 but does appear to control nit-3 gene expression. Unlike nmr+ expression, nit-3 expression occurred to some degree even under nitrogen repression conditions in nmr mutant cells. In wild-type cells, nitrate reductase gene expression was dependent upon the presence of nitrate as an inducer. In sharp contrast, nit-3 mRNA expression occurred to a full extent in three different nit-3 mutants, even in the complete absence of any added inducer. Similarly, a nit-1 mutant which was devoid of nitrate reductase activity because it lacked an essential molybdenum cofactor expressed nit-3 without a requirement for induction by nitrate. These results suggest that nitrate reductase autogenously regulates its own expression and that this control is exerted at the transcriptional level.

Cloning, Molecular↗
PubMed1987

Molecular cloning and analysis of the regulation of nit-3, the structural gene for nitrate reductase in Neurospora crassa.

The nit-3 gene of Neurospora crassa encodes the enzyme nitrate reductase and is regulated by nitrogen catabolite repression and by specific induction with nitrate. The nit-3 gene was isolated from a cosmid-based genomic library by dual selection for benomyl resistance and for the ability to complement a nit-3 mutant strain using the sibling-selection procedure. The nit-3 gene was subcloned as a 3.8-kilobase DNA fragment from a cosmid that carried an approximately 40-kilobase N. crassa DNA insert. A restriction fragment length polymorphism analysis revealed that the cloned segment displayed tight linkage to two linkage-group-4 markers that flank the genomic location of nit-3. RNA gel blot analyses of RNA from wild-type and various mutant strains were carried out to examine the molecular mechanism for regulation of nit-3 gene expression. The nit-3 gene was transcribed to give a large mRNA of approximately 3.4 kilobases, the expected size to encode nitrate reductase. The nit-3 gene was only expressed in wild-type cells subject to simultaneous nitrogen derepression and nitrate induction. A mutant of nit-2, the major nitrogen regulatory gene of Neurospora, did not have detectable levels of nit-3 gene transcripts under the exact conditions in which these transcripts were highly expressed in wild type. Similarly, a mutant of nit-4, which defines a minor positive-acting nitrogen control gene, failed to express detectable levels of the nit-3 transcript. Nitrate reductase gene expression was partially resistant to nitrogen repression in a mutant of the nmr gene, which appears to be a regulatory gene with a direct role in nitrogen catabolite repression. Results are presented that suggest that the enzyme glutamine synthetase does not serve any regulatory role in controlling nitrate reductase gene expression.

Cloning, Molecular↗
PubMed1987

Characterization of nit-2, the major nitrogen regulatory gene of Neurospora crassa.

The nit-2 gene is the major nitrogen-regulatory gene of Neurospora crassa, and under conditions of nitrogen limitations, it turns on the expression of various unlinked structural genes which specify nitrogen-catabolic enzymes. The nit-2 gene was subcloned as a 6-kilobase (kb) DNA fragment from a cosmid that carried approximately a 40-kb N. crassa DNA insert. The nit-2 gene was localized in a DNA segment of approximately 3.5 kb and was shown to correspond to a unique DNA sequence located on linkage group 1. Several N. crassa nit-2 transformants were characterized and were found to possess significantly different levels of the regulated enzyme nitrate reductase. Northern blot analysis of RNA from various strains was carried out to determine whether the nit-2 gene was expressed constitutively or was itself subject to regulation. The results revealed that the nit-2 gene is transcribed to give a single large mRNA of approximately 3.5 kb. Expression of the nit-2 gene is regulated such that its transcript is present at a substantially higher level in cells which are limited for nitrogen than in cells growing under nitrogen-repressed conditions. However, the nit-2 gene is not controlled by autogenous regulation. The nit-2 gene was transcribed only at a low level in nmr-1 and in gln-1b, under both nitrogen-repressed and derepressed conditions, suggesting that these unlinked loci may exert a positive regulatory effect on nit-2.

DNA Restriction Enzymes↗
PubMed1987

Ethanol effects on phospholipids and triacylglycerols of rat brain astrocyte cell line.

A homotypic population of rat brain astrocyte (RBA-1) cell line was used to investigate the effects of ethanol on uptake of [3H]-arachidonic acid and incorporation of label into phospholipids and neutral lipids in the cell. Incubation of the cells with [3H]-arachidonic acid resulted in labeling of most phospholipids and triacylglycerols, although individual phospholipids exhibited different rates for acquiring the label. Labeled arachidonate was first incorporated into phosphatidylcholines and phosphatidylinositols prior to equilibriation with other phospholipids. In short term incubation (8 hr), ethanol (128-255 mM) enhanced the incorporation of labeled arachidonate into most phospholipids. In addition, ethanol at 128 mM enhanced the labeling of the triacylglycerols, and the difference was greater with increasing time of ethanol exposure. It is concluded that ethanol enhanced the uptake and incorporation of polyunsaturated fatty acids into the cell membrane lipids. This study further indicates the feasibility of using the RBA-1 cells in culture to probe the effects of ethanol on membrane lipid metabolism.

Animals↗
PubMedDate not supplied

Intragenic probe used for diagnostics in fragile X families.

The intragenic (FMR-1) probe pE5.1 was used for DNA analysis in fragile X families. With this probe fragments of altered size can be detected in female carriers, affected individuals and transmitting males. The length of the altered fragments was found to vary from one generation to another as well as between sibs. This instability of the DNA detected by pE5.1 was also seen in peripheral blood within single individuals. These phenomena are illustrated by 4 exemplary families segregating the fragile X syndrome. We demonstrate the diagnostic contribution of intragenic analysis to carrier detection as well as the identification of normal transmitting males carrying premutations. One of the families illustrates the passage of a premutation to a male through 2 generations.

DNA↗