Peter J. Russell

A new senescence-inducing mitochondrial linear plasmid in field-isolated Neurospora crassa strains from India

SummarySeveral field-collected strains of Neurospora crassa from the vicinity or Aarey, Bombay, India, are prone to precocious senescence and death. Analysis of one strain, Aarely-1e, demonstrated that the genetic determinants for the predisposition to senescence are maternally inherited. The senescence-prone strains contain a 7-kb, linear, mitochondrial DNA plasmid, maranhar, which is not present in long-lived isolates from the same geographical location. The maranhar plasmid has inverted terminal repeats with protein covalently bound at the 5′ termini. Molecular hybridization experiments have demonstrated no substantial DNA sequence homology between the plasmid and the normal mitochondrial (mtDNA) and nuclear genomes of long-lived strains of N. crassa. Integrated maranhar sequences were detected in the mtDNAs of two cultures derived from Aarey-1e, and mtDNAs with the insertion sequences accumulated during subculturing. Nucleotide sequence analysis of cloned fragments of the two insertion sequences demonstrates that that they are flanked by long inverted repeats of mtDNA. The senescence syndrome of the maranhar strains, and the mode of integration of the plasmid, are reminiscent of those seen in the kalilo strains of N. intermedia. Nonetheless, there is no detectable nucleotide sequence homology between the maranhar and kalilo plasmids.

Regulation of ribosomal RNA cistron number in a strain of Neurospora crassa with a duplication of the nucleolus organizer region

Some progeny from a cross of the translocation mutant T(VL leads to IVL)AR33 with wild-type Neurospora crassa are double nucleolus organizer (DNO) strains, usually displaying two distinct nucleolus organizer regions. The DNO strain is sterile but displays the same growth response as normal laboratory strains of Neurospora. We used DNA-DNA hybridization techniques to quantify the number of rRNA cistrons in the DNO mutant and its vegetative progeny. Comparisons of the rate of hybridization of genomic DNA from the parental AR33 strain and from the DNO strain showed that hybridization was more rapid for the DNO strain than for the parental strain. Successive vegetative progeny of the DNO strain displayed hybridization rates intermediate to those of the original DNO strain and the parental single nucleolus strain, indicating that the number of rRNA cistrons had decreased during vegetative propagation. Estimates of rRNA cistron number obtained from comparisons of the amount of single copy DNA and rDNA hybridized to genomic DNO and AR33 DNA at saturation indicate that the parental AR33 strain contains 225 copies of the rRNA repeat unit while the DNO strain has approx. 440 copies. The number of rRNA cistrons decreases gradually in the successive vegetative progeny, approximating the parental haploid value by the eleventh vegetative transfer.

Magnification of rRNA gene number in a Neurospora crassa strain with a partial deletion of the nucleolus organizer

Some progeny from crosses between the Neurospora crassa translocation strain T(IL→ VL)OY321 and normal sequence N. crassa strains are duplication strains with a partial deletion of the nucleolus organizer. Despite the deletion, these progeny are viable and produce a functional nucleolus. Quantification of rRNA gene number in these deletion progeny demonstrated a significant loss of rRNA genes, down to 60% of the parental wild-type level. Initially, all of these reduced nucleolus organizer (RNO) strains demonstrated a reduction in the rate of mycelial elongation in growth tubes. After several vegetative growth cycles some progeny reverted to the normal growth phenotype, and also showed an increase in the number of rRNA genes to approximately that of the wild type.

Methods for DNA extraction from Candida albicans.

Three different methods are described for the extraction of total genomic DNA from the dimorphic fungus Candida albicans. One method, which enables a large number of cultures to be processed simultaneously, involves pulverizing dried cells with glass beads and then allowing the disrupted cells to break apart, autolyse, by incubation in a solution which includes sorbitol and a nonionic detergent. DNA extraction by a second method with a French pressure cell can be utilized on cultures in any phase of growth, but is not practical for processing numerous samples. The third method, which involves induction of spheroplasts, is commonly used for DNA extraction from various yeasts but is not suited for processing many samples simultaneously. The DNA extracted with the three procedures is comparable in quality; in particular, it is of high molecular size (greater than 30 kbp) and reacts readily with DNA-modifying enzymes such as restriction endonucleases.

Ribosomal DNA inheritance and recombination in Neurospora crassa.

SummaryThe genetic segregation of ribosomal DNA (rDNA) in Neurospora crassa was analyzed by exploiting restriction fragment length polymorphisms in the nontranscribed spacer (NTS) sequences of nine laboratory wild-type strains and wild-collected strains. In an analysis of random spore progeny from seven crosses, and of ordered tetrads from two of those crosses the rDNA was shown to be inherited in a simple, stable Mendelian fashion, exhibiting an approximately 1:1 ratio of the two parental rDNA types. No meiotic recombinants were detected among the progeny, indicating that non-sister-chromatid crossing over is highly suppressed in the rDNA region. The basis for this suppression of meiotic recombination is not known.

Segregation of heterogeneous rDNA segments during demagnification of a Neurospora crassa strain possessing a double nucleolar organizer

SummaryWe have produced a duplication strain of Neurospora crassa, DpAR33-PR-6, which contains two cytologically visible nucleoli (a DNO or double nucleolar organizer strain). When freshly generated, this strain has approximately twice the number of rRNA cistrons found in the parental (single nucleolar organizer) strains. After several serial propagations, there is a marked reduction in rRNA cistron number, approximating that of the SNO parental strains. This reduction in rRNA cistrons (“demagnification”) was not achieved by breakdown of the VL→IVL translocation used to generate the duplication, as rDNA from the two parents can be distinguished by the size of the non-transcribed spacer region in the rDNA repeat unit of each strain. rDNA characteristic of both parents is present even after demagnification, in approximately equal amounts, suggesting the rRNA cistrons are lost randomly and non-preferentially from each homologous chromatid. In addition, the steady-state growth rate appears to be affected by rRNA cistron number, decreasing in freshly generated DNO strains relative to the parental strains, and returning to parental levels after demagnification.

A cold-sensitive mutant ofNeurospora crassa obtained using tritium-suicide enrichment that is conditionally defective in the biosynthesis of cytoplasmic ribosomes

The purpose of this investigation was to use tritium-suicide enrichment with a mutagenized population of wild-typeNeurospora crassa to isolate cold-sensitive mutants with conditional defects in the production of cytoplasmic ribosomes. Eighty-six cold-sensitive mutant strains were obtained following tritium-suicide enrichment using [5-3H]uridine. Zone sedimentation analysis of cytoplasmic ribosomes produced by the strains at 10°C (the nonpermissive temperature) indicated that one strain,PJ31562, is defective in the accumulation of ribosomal subunits at that temperature. The properties of strainPJ31562 are: (1) At 10°C the growth rate is 28 times slower than at 25°C, whereas the factor for the wild type is 5.1. At 25°C the mutants growth rate is 90% that of the wild type. (2) At 10°C the mutant accumulates the two ribosomal subunits, 60 and 37 S, in markedly disproportionate amounts apparently as a result of the underproduction of, or an instability of, the 17 S ribosomal RNA component of the small ribosomal subunit. At 25°C the mutant strain still exhibits a disproportionality in ribosomal subunit accumulation but to a much lesser degree than at 10°C. (3) Genetic studies have shown that a single nuclear gene is responsible for both the cold sensitivity and ribosome biosynthesis defect of strainPJ31562. The mutation involved is located in linkage group IV and appears to be closely linked to, and not allelic with, the cold-sensitive mutation carried by strainPJ30201 which has been shown previously to exhibit a similar phenotype with respect to ribosomal subunit accumulation, and which defines thecrib-1 locus. Thus tritium-suicide enrichment can be used to isolate cold-sensitive mutants ofNeurospora among which a relatively low frequency have conditional defects in ribosome production.

Preserved polyadenylated ribonucleic acid in dormant conidia of Neurospora crassa and new RNA synthesis during spore germination

Molecular hybridization with cloned cDNAs established that transcripts for specific proteins were present in the RNA of dormant conidiospores of Neurospora crassa. Among these were transcripts for the proteolipid subunit of the mitochondrial ATPase-ATP synthase; a larger proportion of the proteolipid transcripts was present in the nonpolyadenylated rather than the polyadenylated RNA fraction (relative to the pattern of germinated spores). The mRNA encoding the Neurospora Mr 83,000 heat-shock protein was also preserved in the dormant spores; however, the transcript for the Mr 30,000 heat-shock protein was not detectable. The polyadenylated RNA from dormant conidia was translatable in vitro; specific immunoprecipitation and electrophoresis assays showed that this preserved RNA included transcripts for nucleus-encoded subunit peptides of the mitochondrial enzymes cytochrome c oxidase and the ATPase-ATP synthase. As determined by molecular hybridization, conidia began to accumulate RNA for the ATPase proteolipid subunit immediately upon their suspension in incubation medium, with a doubling of the quantity of these specific transcripts by 15 min. Total RNA content of the spores dropped sharply during the first 30 min of germination, but by 45 min the amount of total RNA began to increase. Two inhibitors of RNA synthesis, lomofungin and proflavine-SO4, inhibited spore germination by about 90%, in addition to inhibiting incorporation of [3H]uridine into RNA. This inhibition of spore germination by proflavine-SO4 required that the spores be exposed to the drug during the first 60 min after inoculation into nutrient medium; if the inhibitor was added after 60 min of incubation, the spores germinated with normal kinetics. Dormant conidia, therefore, contain a population of preserved mRNA, but the translation of this preserved mRNA is not sufficient for spore germination; new gene transcription appears to be required for the resumption of protein synthesis and germ tube emergence.

GELYSIS: Pascal-implemented analysis of one-dimensional electrophoresis gels.

GELYSIS (GEL analYSIS) is a Pascal implementation of an algorithm that takes data on the distances migrated by DNA fragments in a one-dimension electrophoretic gel and calculates the sizes of the fragments based on the known sizes of marker fragments. A key step in the overall algorithm is a cubic-spline best-fit of marker calibration data. An option of the program is the generation of graphic representations of the gel and of the calibration spline that are convenient for primary data storage or for publication. Provision is made for specifying different band intensities in the graphic output of the gel.

Cloning, Sequencing and Expression of a Full‐Length cDNA Copy of the M1 Double‐Stranded RNA Virus from the Yeast, Saccharomyces cerevisiae

Strains of the budding yeast, Saccharomyces cerevisiae, may contain one or more cytoplasmic viruses with double‐stranded RNA (dsRNA) genomes. The killer phenomenon in yeast, in which one cell secretes a killer toxin that is lethal to another cell, is dependent upon the presence of the L‐A and M1 dsRNA viruses. The L‐A viral genome encodes proteins for the viral capsid, and for synthesis and encapsidation of single‐stranded RNA replication cycle intermediates. The M1 virus depends upon the L‐A‐encoded proteins for its capsid and for the replication of its killer‐toxin‐encoding genome. A full‐length cDNA clone of an M1 genome has been made from a single dsRNA molecule and shown to encode functional killer and killer‐immunity functions. The sequence of the clone indicates minor differences from previously published sequences of parts of the M1 genome and of the complete genome of S14 (an internal deletion derivative of M1) but no unreported amino acid variants and no changes in putative secondary structures of the single‐stranded RNA. A 118‐nucleotide contiguous segment of the M1 genome has not previously been reported; 92 of those nucleotides comprise a segment of A nucleotides in the AU‐rich bubble that follows the toxin‐encoding reading frame. The GenBank Accession Number for the sequence is U78817; the locus is SCU78817.