Biomedical subjects

Y H Fu

Publications and source records attributed to Y H Fu.

At least 55 records · Page 3Linked to original sources

PubMed1991

Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome.

Fragile X syndrome is the most frequent form of inherited mental retardation and is associated with a fragile site at Xq27.3. We identified human YAC clones that span fragile X site-induced translocation breakpoints coincident with the fragile X site. A gene (FMR-1) was identified within a four cosmid contig of YAC DNA that expresses a 4.8 kb message in human brain. Within a 7.4 kb EcoRI genomic fragment, containing FMR-1 exonic sequences distal to a CpG island previously shown to be hypermethylated in fragile X patients, is a fragile X site-induced breakpoint cluster region that exhibits length variation in fragile X chromosomes. This fragment contains a lengthy CGG repeat that is 250 bp distal of the CpG island and maps within a FMR-1 exon. Localization of the brain-expressed FMR-1 gene to this EcoRI fragment suggests the involvement of this gene in the phenotypic expression of the fragile X syndrome.

Alleles↗

PubMed1991

Nucleotide sequence, messenger RNA stability, and DNA recognition elements of cys-14, the structural gene for sulfate permease II in Neurospora crassa.

The complete nucleotide sequence of the cys-14 gene which encodes sulfate permease II, a member of the sulfur regulatory circuit, is presented. The cys-14 gene contains four introns with consensus splice site sequences and is transcribed from four closely spaced initiation sites located approximately 20 bp upstream of the ATG initiation codon. The translated CYS14 protein is composed of 781 amino acids with a molecular weight of 87,037 and contains 12 potential hydrophobic membrane-spanning domains. cys-4 mRNA was found to turn over with a half-life of approximately 15 min, which presumably contributes to the regulation of sulfate permease II function. The cys-14 gene is highly expressed, but only in cells subject to sulfur limitation, and is turned on by the positive-acting CYS3 sulfur regulatory protein. Results are presented which show that CYS3 protein binds with higher affinity to DNA fragments which contain two or three tandem copies of a binding site sequence. Analyses of binding site specificity via mutated binding site elements showed that different regions of the partially symmetrical CYS3 binding site are important for recognition by the CYS3 regulatory protein.

Amino Acid Sequence↗

PubMed1991

Nit-3, the structural gene of nitrate reductase in Neurospora crassa: nucleotide sequence and regulation of mRNA synthesis and turnover.

The nit-3 gene of the filamentous fungus Neurospora crassa encodes the enzyme nitrate reductase, which catalyzes the first reductive step in the highly regulated nitrate assimilatory pathway. The nucleotide sequence of nit-3 was determined and translates to a protein of 982 amino acid residues with a molecular weight of approximately 108 kDa. Comparison of the deduced nit-3 protein sequence with the nitrate reductase protein sequences of other fungi and higher plants revealed that a significant amount of homology exists, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The synthesis and turnover of the nit-3 mRNA were also examined and found to occur rapidly and efficiently under changing metabolic conditions.

Amino Acid Sequence↗

PubMed1991

Deletion analysis of the F plasmid oriT locus.

Functional domains of the Escherichia coli F plasmid oriT locus were identified by deletion analysis. DNA sequences required for nicking or transfer were revealed by cloning deleted segments of oriT into otherwise nonmobilizable pUC8 vectors and testing for their ability to promote transfer or to be nicked when tra operon functions were provided in trans. Removal of DNA sequences to the right of the central A + T-rich region (i.e., from the direction of traM) did not affect the susceptibility of oriT to nicking functions; however, transfer efficiency for oriT segments deleted from the right was progressively reduced over an 80- to 100-bp interval. Deletions extending toward the oriT nick site from the left did not affect the frequency of transfer if deletion endpoints lay at least 22 bp away from the nick site. Deletions or insertions in the central, A + T-rich region caused periodic variation in transfer efficiency, indicating that phase relationships between nicking and transfer domains of oriT must be preserved for full oriT function. These data show that the F oriT locus is extensive, with domains that individually contribute to transfer, nicking, and overall structure.

Base Composition↗

PubMed1991

nit-4, a pathway-specific regulatory gene of Neurospora crassa, encodes a protein with a putative binuclear zinc DNA-binding domain.

nit-4, a pathway-specific regulatory gene in the nitrogen circuit of Neurospora crassa, is required for the expression of nit-3 and nit-6, the structural genes which encode nitrate and nitrite reductase, respectively. The complete nucleotide sequence of the nit-4 gene has been determined. The predicted NIT4 protein contains 1,090 amino acids and appears to possess a single Zn(II)2Cys6 binuclear-type zinc finger, which may mediate DNA binding. Site-directed mutagenesis studies demonstrated that cysteine and other conserved amino acid residues in this possible DNA-binding domain are necessary for nit-4 function. A stretch of 27 glutamines, encoded by a CAGCAA repeating sequence, occurs in the C terminus of the NIT4 protein, and a second glutamine-rich domain occurs further upstream. A NIT4 protein deleted for the polyglutamine region was still functional in vivo. However, nit-4 function was abolished when both the polyglutamine region and the glutamine-rich domain were deleted, suggesting that the glutamine-rich domain might function in transcriptional activation. The homologous regulatory gene from Aspergillus nidulans, nirA, encodes a protein whose amino-terminal half has approximately 60% amino acid identity with NIT4 but whose carboxy terminus is completely different. A hybrid nit-4-nirA gene was constructed and found to function in N. crassa.

Amino Acid Sequence↗

PubMed1990

Nucleotide sequence and DNA recognition elements of alc, the structural gene which encodes allantoicase, a purine catabolic enzyme of Neurospora crassa.

The nitrogen regulatory circuit of Neurospora crassa contains structural genes that encode nitrogen catabolic enzymes which are subject to complex genetic and metabolic regulation. This set of genes is controlled by nitrogen limitation, by specific induction, and by the action of nit-2, a major positive-acting regulatory gene, and nmr, a negative-acting control gene. The complete nucleotide sequence of alc, the gene that encodes allantoicase, a purine catabolic enzyme, is presented. The alc gene contains a single intron, is transcribed from two initiation sites situated approximately 50 nb upstream of the translation start site, and encodes a protein comprised of 354 amino acids. Mobility shift and DNA footprint experiments identified a single binding site for the NIT2 regulatory protein in the alc promoter region. The binding site contains a 10 nucleotide base pair symmetrical sequence which is flanked by two possible core binding sequences, TATCT and TATCG. Mutant NIT2/beta-gal fusion proteins with amino acid substitutions in a putative zinc-finger motif were shown to be completely deficient in the ability to bind to the alc promoter DNA fragment.

Amino Acid Sequence↗

PubMed1990

cys-3, the positive-acting sulfur regulatory gene of Neurospora crassa, encodes a sequence-specific DNA-binding protein.

cys-3, the positive-acting master sulfur regulatory gene of Neurospora crassa, turns on the expression of an entire set of unlinked structural genes which encode sulfur-catabolic enzymes. cys-3 encodes a protein of 236 amino acid residues and contains a potential bipartite DNA-binding domain which consists of a leucine zipper and an adjacent highly basic region. Gel band mobility shift and DNA footprint experiments were used to demonstrate that the CYS3 protein, expressed in Escherichia coli, binds to three distinct sites in the 5' upstream DNA of cys-14, the structural gene for sulfate permease II. The CYS3 protein also binds to one distinct sequence element upstream of the cys-3 gene itself, which suggests an autoregulatory role for this protein. Two mutant CYS3 proteins, altered in the basic region of the DNA-binding domain, failed to bind to either the cys-14 or the cys-3 upstream recognition elements.

Amino Acid Sequence↗

PubMed1990

Regulation of branched-chain amino acid biosynthesis in Neurospora crassa: cloning and characterization of the leu-1 and ilv-3 genes.

The genes coding for the branched-chain amino acid biosynthetic enzymes comprise an integrated regulatory system. The expression of the several structural genes coding for enzymes of the isoleucine-valine and leucine pathways is controlled in parallel by the positive-acting regulatory gene, leu-3. The leu-1 and ilv-3 genes, coding for beta-isopropyl-malate dehydrogenase and aceto-hydroxyacid synthase, respectively, were cloned from a cosmid library. Restriction fragment length polymorphism analysis revealed that the two cloned fragments indeed mapped to the genomic locations of the leu-1 and ilv-3 genes, respectively. Northern blot analysis demonstrated that the leu-1 gene is transcribed to give an mRNA of approximately 1.5 kb, whereas the ilv-3 transcript size is 2.6 kb. The expression of both genes appears to be regulated at the transcriptional level. One leu-3 regulatory mutant was greatly deficient in both leu-1 and ilv-3 mRNAs, whereas another leu-3 allele showed an unusual antiparallel pattern of regulation.

3-Isopropylmalate Dehydrogenase↗

PubMed1990

Nucleotide sequence and analysis of NMR, a negative-acting regulatory gene in the nitrogen circuit of Neurospora crassa.

In Neurospora the expression of a set of unlinked structural genes, which allows utilization of various nitrogen-containing compounds, is controlled by the positive-acting nit-2 gene and the negative-acting nmr gene. The nucleotide sequence of the nmr gene has been determined and a long open reading frame which encodes a putative protein of 54854 daltons has been identified. A full-length cDNA clone was obtained and its the sequence revealed that the nmr gene contains no introns. The transcriptional start and stop sites have been mapped by S1 nuclease and primer extension. Site-directed mutagenesis was used to introduce stop codons at various locations in the nmr coding region. Transformation assays showed that the proteins lacking up to 16% of the carboxyl-terminus were still functional. Homology searches showed that the nmr protein is homologous to the yeast arginine regulatory gene AR-GRII.

Amino Acid Sequence↗

PubMed1990

Molecular cloning and characterization of alc the gene encoding allantoicase of Neurospora crassa.

Purines can be utilized as a secondary nitrogen source by Neurospora crassa during conditions of nitrogen limitation. The expression of purine catabolic enzymes is governed by the nitrogen regulatory circuit and requires induction by uric acid. The major positive-acting nitrogen regulatory gene, nit-2, turns on the expression of the purine catabolic enzymes, which may also be subject to negative regulation by a second control gene, nmr. We have cloned alc, the structural gene which encodes allantoicase, an inducible enzyme of the purine degradative pathway. The identity of the alc clone was confirmed by restriction fragment length polymorphism analysis and by repeat-induced mutation. The alc gene is transcribed to give a single messenger RNA, approximately 1.2 kb in length. The negative-acting nmr gene affects the expression of alc in the expected manner. Both the nit-2 and the nmr control genes affect alc mRNA levels and allantoicase enzyme activity in both the induced and nitrogen-repressed conditions.

Blotting, Northern↗

PubMed1990

nit-2, the major positive-acting nitrogen regulatory gene of Neurospora crassa, encodes a sequence-specific DNA-binding protein.

The nit-2 major nitrogen regulatory gene of Neurospora crassa turns on the expression of various unlinked structural genes that specify nitrogen-catabolic enzymes under nitrogen-limitation conditions. The nit-2 gene encodes a protein of 1036 amino acid residues with a single zinc finger and a downstream basic region that may make up a DNA-binding domain. The zinc-finger domain of the NIT2 protein was synthesized in two ways to examine its DNA-binding activity with gel-band-mobility shift and DNA-footprint experiments. The NIT2 protein binds to specific DNA recognition elements that are located upstream of nitrogen-regulated structural genes. Each recognition element contains at least two copies of a core sequence whose consensus is TATCTA.

Base Sequence↗

PubMed1990

Site-directed mutagenesis of the 'zinc finger' DNA-binding domain of the nitrogen-regulatory protein NIT2 of Neurospora.

The major nitrogen-regulatory gene nit-2 of Neurospora crassa activates the expression of numerous unlinked structural genes which specify nitrogen-catabolic enzymes during conditions of nitrogen limitation. The nit-2 gene encodes a regulatory protein of 1036 amino acid residues with a single 'zinc finger' and a downstream basic region, which together may constitute a DNA-binding domain. The zinc finger domain of the NIT2 protein was synthesized in vitro and also expressed as a fusion protein in Escherichia coli to examine its DNA-binding activity. The wild-type NIT2 finger domain protein binds to the promoter region of nit-3, the nitrate reductase structural gene. A series of NIT2 mutant proteins obtained by site-directed mutagenesis was expressed and tested for functional activity. The results demonstrate that both the single zinc-finger motif and the downstream basic region of NIT2 are critical for its trans-activating function in vivo and specific DNA-binding in vitro.

Amino Acid Sequence↗

PubMed1990

Intrinsic bends and integration host factor binding at F plasmid oriT.

F plasmid oriT DNA extending from the F kilobase coordinate 66.7 (base pair [bp] 1 on the oriT sequence map) rightward to bp 527 was analyzed for intrinsic bends (by permutation assays) and for binding of integration host factor (IHF) (by gel retardation and DNase footprinting). Intrinsic bending of the 527-bp fragment (bend center approximately at bp 240) was represented as a composite of at least two components located near bp 170 and near bp 260. IHF bound primarily to a site extending from bp 165 to 195 and with lower affinity to a site extending from bp 287 to 319. The intrinsic curvature and sequences to which IHF binds (IHF is known to bend DNA) may play a structural role in oriT function.

Bacterial Proteins↗

PubMed1990

nit-2, the major nitrogen regulatory gene of Neurospora crassa, encodes a protein with a putative zinc finger DNA-binding domain.

The nitrogen regulatory circuit of Neurospora crassa consists of a set of unlinked structural genes which specify various nitrogen catabolic enzymes plus control genes and metabolic effectors which regulate their expression. The positive-acting nit-2 regulatory gene is required to turn on the expression of the nitrogen catabolic enzymes during conditions of nitrogen limitation. The complete nucleotide sequence of the nit-2 gene was determined. The nit-2 mRNA is 4.3 kilobases long and has a long nontranslated sequence at both its 5' and 3' ends. The nit-2 gene nucleotide sequence can be translated to yield a protein containing 1,036 amino acid residues with a molecular weight of approximately 110,000. Deletion analyses demonstrated that approximately 21% of the NIT2 protein at its carboxy terminus can be removed without loss of function. The nit-2 protein contains a single putative Cys2/Cys2 zinc finger domain which appears to function in DNA binding and which has striking homology to a mammalian trans-acting factor, GF-1.

Amino Acid Sequence↗

PubMed1989

Isolation of nit-4, the minor nitrogen regulatory gene which mediates nitrate induction in Neurospora crassa.

Expression of nitrate reductase in Neurospora crassa requires the positive action of nit-4, a pathway-specific regulatory gene, which mediates nitrate induction. We report the molecular cloning of the nit-4 gene and present results which suggest that the nit-4 gene is constitutively expressed to yield a low-abundance 2.2-kilobase transcript. These results indicate that the nit-2 major control gene and the nit-4 pathway-specific control gene independently regulate the expression of the nitrate assimilatory structural genes.

Cloning, Molecular↗

PubMed1989

cys-3, the positive-acting sulfur regulatory gene of Neurospora crassa, encodes a protein with a putative leucine zipper DNA-binding element.

The sulfur-regulatory circuit of Neurospora crassa consists of a set of unlinked structural genes which encode sulfur-catabolic enzymes and two major regulatory genes which govern their expression. The positive-acting cys-3 regulatory gene is required to turn on the expression of the sulfur-related enzymes, whereas the other regulatory gene, scon, acts in a negative fashion to repress the synthesis of the same set of enzymes. Expression of the cys-3 regulatory gene was found to be controlled by scon and by sulfur availability. The nucleotide sequence of the cys-3 gene was determined and can be translated to yield a protein of molecular weight 25,892 which displays significant homology with the oncogene protein Fos, yeast GCN4 protein, and sea urchin histone H1. Moreover, the putative cys-3 protein has a well-defined leucine zipper element plus an adjacent charged region which together may make up a DNA-binding site. A cys-3 mutant and a cys-3 temperature-sensitive mutant lead to substitutions of glutamine for basic amino acids within the charged region and thus may alter DNA-binding properties of the cys-3 protein.

Amino Acid Sequence↗

PubMed1989

[Serum copper levels and pathologic changes in the fetal membranes in cases of premature rupture of the membranes].

140 samples of serum and amnionic membrane collected from 60 patients with premature rupture of membranes (PROM) and 20 controls were assayed for levels of essential trace elements by inductively coupled plasma atomic emission spectrometry (ICPAES). 72 samples of amnionic membranes were examined by pathologist with HE. Van Gieson's and Wright's strain. The serum copper level in PROM cases was significantly lower (23.438 8 +/- 3.292 4 mumol/L) as compared with that of the controls (30.044 1 +/- 4.416 1 mumol/L), P less than 0.01. The serum and tissue level of essential trace elements (Cr, Mn) showed no difference. The collagen and elastin in the amnionic membranes were sparse and weak. It is pointed out that the low copper level and the sparse and weak collagen and elastin in amnionic membranes may be related to PROM.

Copper↗

PubMed1988

Molecular cloning and characterization of a negative-acting nitrogen regulatory gene of Neurospora crassa.

Expression of the structural genes of the nitrogen control circuit of Neurospora crassa is regulated by the positive-acting nit-2 control gene and by the negative-acting nmr control gene. Nitrate reductase is expressed in a constitutive fashion in nmr mutant strains, which appear to be largely insensitive to nitrogen catabolite repression. Thus, nmr mutants are sensitive to chlorate in the presence of ammonia or glutamine, whereas the wild type is chlorate resistant under these conditions. A cosmid library was screened for the presence of the nmr+ gene by the sib selection procedure, and a single cosmid was isolated which transforms the nmr mutant to chlorate resistance at a high frequency. A restriction fragment length polymorphism analysis revealed that the cloned DNA segment maps to the precise genomic location of nmr. Northern blot analyses revealed that the nmr gene is itself not regulated but is expressed constitutively to give a single transcript of approximately 1.8 kb.

Chlorates↗