Search

In the ascomycete Leptosphaeria maculans tandem repeats of ribosomal DNA (rDNA) are restricted to one or two particular chromosomes of the 15 chromosomes of 19 field isolates examined. Ribosomal DNA can account for size differences of 35% between homologous chromosomes in a particular tetrad. During crossing, no detectable recombination between blocks of tandem repeats, nor changes in their size occur. The organisation of rDNA in L. maculans differs from many other haploid fungi. Firstly, sequence heterogeneity occurs within tandem repeats of rDNA; regularly spaced Sal 1 sites (0.25 Mb apart) are present within a 1.4 Mb block of tandem repeats. Secondly, individual isolates have different-sized rDNA repeats; this variation occurs in the non-transcribed intergenic spacer region. Thirdly, there is a wide range in the copy number of the rDNA repeat (from 56 to 225) amongst only four field isolates examined.

Pulsed field gel electrophoresis (PFGE), or electrophoretic karyotyping, separates chromosomal-sized pieces of DNA in agarose gels where the orientation of the electric field is periodically altered. This technique has revealed that many fungi have a high degree of chromosomal length polymorphisms. Often the only isolates with identical karyotypes are derived from a single clone, thus PFGE provides a 'genetic fingerprint' for them. The size range and number of chromosomes within isolates of a particular species are usually constant, hence PFGE can distinguish between morphologically similar fungi. This technique can also be used to follow inheritance of chromosomal length polymorphisms and shows that in some fungi novel-sized chromosomes are produced during meiosis. As well as resolving the nuclear (A-type) chromosomes, it can also resolve dispensable (B-type) chromosomes and cytoplasmic genomes including mitochondrial DNA and linear plasmids. The application of this technique to Australian isolates of Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus), is discussed.

Pulsed field gel electrophoresis experiments show that chromosomal length polymorphisms are produced during meiosis in the ascomycetous plant pathogen Leptosphaeria maculans. Homologues in tetrads of L. maculans were identified on the basis of their binding to chromosome-specific probes that included beta-tubulin, nitrate reductase, 18S ribosomal DNA and two Random Amplified Polymorphic DNA (RAPD) markers. Changes in size of homologues were followed during meiosis. Significant karyotype variation was evident due to the random assortment of parental homologues of different sizes. In most cases, the progeny had the same-sized homologues as the parents; however, in some instances novel-sized homologues were detected that varied in size from those of the parents by up to 50 kb. Our results are consistent with the hypothesis that these novel chromosomal length polymorphisms are produced by reciprocal recombination between parental homologous chromosomes of unequal sizes.

Southern hybridisation, polymerase chain reaction (PCR), and nucleotide sequence, data indicate that the 5s ribosomal RNA (rRNA) gene is linked to the main rRNA gene repeat in the nuclear genome of four cryptomonad algae (Rhinomonas pauca, Storeatula major, Komma caudata, and isolate Cs 134). The 5s gene is apparently transcribed in the same direction as the large and small subunit rRNA genes. The intergenic spacer between the 5s gene and the large subunit rRNA gene exhibits length and sequence polymorphism among the different species. Cryptomonads contain two different eukaryotic genomes: the host nucleus and the nucleus of a eukaryotic endosymbiont. Mapping experiments with isolated chromosomes of the host and endosymbiont genomes showed that the intergenic spacer between the large subunit and the 5s rRNA gene, which was amplified from total DNA by PCR, was derived from the host nuclear genome.

Two linear DNA plasmids (pLm9 and pLm10, sized 9 and 10 kb respectively) were isolated from the phytopathogenic ascomycete Leptosphaeria maculans, using pulsed-field gel electrophoresis. pLm9 and pLm10 are found only in aggressive isolates of L. maculans but, because aggressive and non-aggressive strains appear to be different species, these plasmids are probably not involved in pathogenicity. pLm9 and pLm10 copurify with a mitochondrially-enriched cell fraction, and do not hybridise to chromosomal or mitochondrial DNA, or to each other. Exonuclease digestions suggest that both these molecules contain covalently-bound proteins at their 5' termini. pLm9 hybridises to the RNA polymerase of a linear plasmid from the ascomycete Podospora anserina, and pLm10 hybridises to the DNA polymerase from the same P. anserina plasmid, suggesting that pLm9 and pLm10 encode their own replication and transcription enzymes.

The nitrate reductase (niaD) gene was isolated from the phytopathogenic loculoascomycete Leptosphaeria maculans by screening a genomic DNA library with the Aspergillus nidulans niaD gene. The L. maculans niaD gene is the first protein-encoding gene characterised from this fungus. It encodes a predicted protein of 893 amino acids and contains four putative introns at positions in the gene equivalent to those of four of the six introns in the A. nidulans niaD gene. Mutants defective in niaD and molybdenum cofactor gene(s) of L. maculans have been isolated. Transformation of a L. maculans niaD mutant with a 3.8 kb SacII fragment containing the L. maculans niaD gene restored wild-type growth on nitrate as a sole nitrogen source. The niaD gene is present as a single copy on a chromosome which ranges in size from 2.6 to 2.8 Mb between the different L. maculans isolates examined.

Chromosomal DNA of Australian field-isolates of the phytopathogenic ascomycete Leptosphaeria maculans was resolved by pulsed-field gel electrophoresis. All isolates examined had highly variable karyotypes. Ascospores (sexual spores) derived from single pseudothecia (sexual fruiting bodies) isolated from Brassica napus (oilseed rape) stubble were analyzed. In two tetrads four distinct karyotypes were observed, with only one chromosomal DNA band in common to all the members of each tetrad. Although isolates had highly variable karyotypes, two overall patterns were present. In one pattern there were at least 12 chromosomal DNA bands, the largest being greater than 2.2 Mb in size; in the other there were more than 15 chromosomal DNA bands, the largest being about 2.0 Mb. The chromosomal DNA preparations included mitochondrial DNA which migrated as a diffuse band between 0.10 and 0.15 Mb in size, and DNA molecules of 8 and 9 kb in size.

Milk production, feed efficiency, health, and reproduction were evaluated in 46 Jersey cows that received either 500 mg of sometribove (n-methionyl bST) in a prolonged-release formulation or an equivalent volume of excipient bi-weekly beginning at 60 +/- 3 DIM. Cows were fed a TMR for ad libitum intake and were milked twice daily. Treatment with sometribove increased 3.5% FCM production 5.3 kg/d (31.4%) over controls. Milk composition was not changed, except that milk P content averaged 51 ppm higher in bST-treated cows. Net energy intake was 4.8 Mcal/d (22.9%) higher in the bST-treated cows than in the controls. General health of all cows was good throughout the study, but the cows treated with bST had more cases of mastitis than did the control cows. The bST treatment produced localized reactions at the site of injection in some cows, but these reactions did not affect milk production. Sometribove treatment had no effect on reproductive performance; 85% of the treated and 100% of the control cows calved successfully. Treatment with bST for a full lactation did not affect performance during the initial 60 d of the subsequent lactation.

Southern hybridization and polymerase chain reaction data indicate that the 5S ribosomal RNA gene is linked to the ribosomal RNA gene repeat unit in the oomycetes, Phytophthora vignae, P. cinnamomi, P. megasperma f.sp. glycinea and Saprolegnia ferax, and is apparently transcribed in the same direction as the large and small subunit ribosomal RNA genes. The polymerase chain reaction has been used to amplify all components of the entire ribosomal RNA gene repeat unit for each of these oomycetes. The total size of all amplified products is identical to the size of the ribosomal RNA gene repeat unit, as determined by Southern analysis.

Addition of dithiothreitol in the Lowry procedure 3 min after adding the Folin-Ciocalteau reagent produces immediate color development, with 35 to 60% greater absorbance per mass of protein used.