Nucleotide sequence of Neurospora crassa cytoplasmic initiator tRNA.

Initiator methionine tRNA from the cytoplasm of Neurospora crassa has been purified and sequenced. The sequence is: pAGCUGCAUm1GGCGCAGCGGAAGCGCM22GCY*GGGCUCAUt6AACCCGGAGm7GU (or D) - CACUCGAUCGm1AAACGAG*UUGCAGCUACCAOH. Similar to initiator tRNAs from the cytoplasm of other eukaryotes, this tRNA also contains the sequence -AUCG- instead of the usual -TphiCG (or A)- found in loop IV of other tRNAs. The sequence of the N. crassa cytoplasmic initiator tRNA is quite different from that of the corresponding mitochondrial initiator tRNA. Comparison of the sequence of N. crassa cytoplasmic initiator tRNA to those of yeast, wheat germ and vertebrate cytoplasmic initiator tRNA indicates that the sequences of the two fungal tRNAs are no more similar to each other than they are to those of other initiator tRNAs.     

Nucleotide sequence of 5S ribosomal RNA from Aspergillus nidulans and Neurospora crassa

The nucleotide sequences of 5S rRNA molecules isolated from the cytosol and the mitochondria of the ascomycetes A. nidulans and N. crassa were determined by partial chemical cleavage of 3'-terminally labelled RNA. The sequence identity of the cytosolic and mitochondrial RNA preparations confirms the absence of mitochondrion-specific 5S rRNA in these fungi. The sequences of the two organisms differ in 35 positions, and each sequence differs from yeast 5S rRNA in 44 positions. Both molecules contain the sequence GCUC in place of GAAC or GAUY found in all other 5S rRNAs, indicating that this region is not universally involved in base-pairing to the invariant GTpsiC sequence of tRNAs.     

Nucleotide sequence of the Erwinia chrysanthemi gene encoding 2-keto-3-deoxygluconate permease

The phytopathogenic bacterium Erwinia chrysanthemi produces a group of pectolytic enzymes able to depolymerise the pectic compounds in plant cell walls. The resulting tissue maceration is known as soft rot disease. The degraded pectin products are transported by 2-keto-3-deoxygluconate permease into the bacterial cell, where they serve as carbon and energy sources. This H+ coupled transport system is encoded by the kdgT gene; we report the nucleotide sequence of kdgT. It is encoded by an open reading frame (ORF) of 1194 bp, which is preceded by an Escherichia coli-type promoter region. The ORF encodes a protein with 398 amino acid (aa) residues and a predicted Mr of 48,550. As would be expected for a membrane protein, it is very hydrophobic, containing 63% nonpolar aa. However, the kdgT gene has no apparent evolutionary relationship to other genes encoding sugar transport proteins, such as lacY, melB or the E. coli citrate transport gene. Southern hybridization experiments indicate a strong homology between the Er. chrysanthemi and E. coli kdgT genes; there is also a second region on the E. coli chromosome with homology to kdgT. The kdgT gene is located near the ade-377 marker on the Er. chrysanthemi chromosome (equivalent to the region between 20 and 30 min in E. coli), whereas the E. coli kdgT gene is located at 88 min. Thus, these two enterobacteria show some significant differences in their genomic organization.     

Nucleotide sequence of a human heart cDNA encoding the mitochondrial phosphate carrier.

We have isolated and characterized a full length cDNA clone encoding the precursor of the human heart mitochondrial phosphate carrier protein. The entire clone is 1330 bp in length with 5'- and 3'-untranslated regions of 48 and 184 bp, respectively. The open reading frame encodes the mature protein consisting of 312 amino acids, preceded by a presequence of 49 amino acids. The amino acid sequence of the mature human phosphate carrier is 93.6, 94.2 and 33.6% identical to that of the phosphate carrier from beef, rat and yeast, respectively. Like other mitochondrial transport proteins, the human phosphate carrier has a tripartite structure. Each of the three repeats contains two hydrophobic regions which presumably span the membrane in the form of alpha-helices.     

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.     

A High-Density Single Nucleotide Polymorphism Map for Neurospora crassa

We report the discovery and validation of a set of single nucleotide polymorphisms (SNPs) between the reference Neurospora crassa strain Oak Ridge and the Mauriceville strain (FGSC 2555), of sufficient density to allow fine mapping of most loci. Sequencing of Mauriceville cDNAs and alignment to the completed genomic sequence of the Oak Ridge strain identified 19,087 putative SNPs. Of these, a subset was validated by cleaved amplified polymorphic sequence (CAPS), a simple and robust PCR-based assay that reliably distinguishes between SNP alleles. Experimental confirmation resulted in the development of 250 CAPS markers distributed evenly over the genome. To demonstrate the applicability of this map, we used bulked segregant analysis followed by interval mapping to locate the csp-1 mutation to a narrow region on LGI. Subsequently, we refined mapping resolution to 74 kbp by developing additional markers, resequenced the candidate gene, NCU02713.3, in the mutant background, and phenocopied the mutation by gene replacement in the WT strain. Together, these techniques demonstrate a generally applicable and straightforward approach for the isolation of novel genes from existing mutants. Data on both putative and validated SNPs are deposited in a customized public database at the Broad Institute, which encourages augmentation by community users.     

Nucleotide sequence and conserved features of the 5.8 S rRNA coding region of Neurospora crassa.

The nucleotide sequence of Neurospora crassa 5.8 S rDNA and adjacent regions has been determined. The deduced 5.8 S rRNA sequence of Neurospora differs from the 5.8 S rRNA sequence of Saccharomyces cerevisiae at 13 of 158 residues. Nine of these differences are clustered in a segment capable of forming a short hairpin secondary structure thought to be involved in the 28 S - 5.8 S rRNA complex. These differences occur in pairs such that the potential secondary structure is preserved.     

Nucleotide sequence of the cloned mRNA and gene of the ADP/ATP carrier from Neurospora crassa.

A cDNA complementary to the mRNA of the ADP/ATP carrier from Neurospora crassa was identified among ordered cDNA clones by hybridizing total polyadenylated RNA to pools of 96 cDNA recombinant plasmids and subsequent cell-free translation of hybridization-selected mRNA. Further carrier cDNAs were found by colony filter hybridization at a frequency of 0.2-0.3%. The gene of the carrier was cloned and isolated on a 4.6-kbp EcoRI fragment of total Neurospora DNA, and the start of the mRNA was determined by S1 nuclease mapping. From the nucleotide sequence of the cDNA and the genomic DNA, the primary structure of the gene, of the mRNA and of the ADP/ATP carrier protein could be deduced. The gene occurs in a single copy in the genome and related genes are absent. It contains two short introns, and a pyrimidine-rich promoter region. The mRNA has a 46-bp 5' end and a 219-bp 3' end. There is an open reading frame coding for the 313 amino acid residues of the Neurospora carrier protein. The amino acid sequence is homologous in 148 positions with the established primary structure of the beef heart carrier.