Ian K. Ross

The polymerase chain reaction and its application to filamentous fungi

The polymerase chain reaction (PCR) is a rapid and sensitive procedure for the in vitro amplification of specific segments of DNA. The ability to perform primer-directed amplification of specific sequences of DNA has had an effect on research similar to that of the discovery of restriction enzymes and Southern hybridization. Since the introduction of PCR in 1986, an ever increasing number of scientific applications have been reported, including the direct cloning, mutagenesis, sequencing and exact engineering of specific genes or gene sequences directly from genomic DNA or complementary DNA (cDNA). PCR is also being used increasingly for a variety of genetic and forensic analysis, as well as for several diagnostic procedures useful in screening for genetic diseases or disease agents. The success of PCR in generating and detecting specific DNA fragments has accelerated the use of molecular biology in many biological systems. The focus of this review is to offer examples of how PCR techniques may be used to study the molecular genetics, life cycles, ecology and phylogeny of filamentous fungi.

Allozymes of a wild Agaricus bisporus population: new alleles, new genotypes

Eighteen field-collected isolates ofAgaricus bisporus from coastal central California and two extra? limital field isolates were subjected to allozyme analysis via PAGE, and were genotypically scored at six polymorphic loci. This population sample carries previously unreported alleles at three of five loci for which a large culture collection (PSUMCC), composed primarily of cultivar lines, had previously been screened. Only one allele known from the PSUMCC collection was not found in the wild popu? lation. Novel combinations of previously known alleles were also found. With one exception, each field isolate was genetically unique within the field sample. The wild population of A. bisporus thus contains a large number of novel genotypes that are not represented in cultivar lines. These facts strongly suggest that A. bisporus is indigenous to North America. Our observations, particularly the discovery of several new alleles, also indicate that the wild population of this species represents a significant reservoir of genetic information presently unexploited by the commercial mushroom industry. In contrast, four of the field-collected isolates could be assigned to genotypic classes known from the PSUMCC collection or other cultivars. These findings support the widely-held belief that cultivars of A. bisporus do escape from commercial cultivation and can reproduce under natural conditions.

The Stemonitomycetidae, A New Subclass of Myxomycetes

In D. disciformis the sporangia are small, 0.3-0.8 mm in diam, and the spores are purple-brown in mass, 11-13 p/ in diam, minutely but densely warted and usually reticulate. In D. vaccinum the sporangia are larger, 0.6-1.0 mm in diam, and the spores are black in mass, 9-12 / in diam, and sparsely warted without any tendency for the warts to be arranged in a reticulum. Finally, D. vaccinum does not have the basal region of the inner peridium persisting as a distinct disk, which is the major feature of D. disciformis.

Ultrastructure of the plasmodial slime mold Perichaena vernicularis. II. Formation of the peridium.

SummaryThe development of the peridium ofPerichaena vermicularis has been examined using light and electron microscopy and acid phosphatase localization. A newly formed fruiting body consists of undifferentiated protoplasm which is enveloped by a slime coat. Almost immediately after formation of the plasmodiocarp, the protoplasm differentiates into autolytic and fruiting regions. The autolytic region is located at irregular intervals between the slime coat and the fruiting region and separated from both of them by membranes.Soon after the autolytic region has formed, additional signs of degeneration appear in the autolytic region including unusual appearance of nuclei, increase in autophagic vacuoles, and the presence of clear areas in the ground substance. The plasma membrane, which once completely separated the slime coat from the autolytic region, is no longer continuous. Electron micrographs of the autolytic region from later developmental stages show formation of extensive channels which contain protoplasm in various stages of degradation. Acid phosphatase is present in the channels of the autolytic region. The morphological evidence and the presence of hydrolytic enzyme suggest the region is being digested and re-adsorbed.After the autolytic region has been digested, an even layer of peridial wall material is laid down, and at regular intervals additional wall material is produced. The additional wall material forms the reticulation on the inside of the peridial wall.

EXTRACELLULAR LACCASES: BIOCHEMICAL MARKERS FOR AGARICUS SYSTEMATICS

Isozyme analysis of extracellular laccases (ECLs) of Agaricus spp. on polyacrylamide gels revealed patterns of activity which were dissimilar between species but largely invariant within species. In many species the various ECL electromorphs in the culture medium appeared and disappeared, or became more or less abundant, in a temporal sequence of several weeks duration. There is a tendency for some ECL bands to increase in mobility by 1-2 percent as cultures age. Electrophoretic comparison of ECLs appears to be a promising method contributing toward systematic resolution and phylogenetic reconstruction in Agaricus.

Ultrastructure of the plasmodial slime moldPerichaena vermicularis

SummaryMature plasmodia ofPerichaena vermicularis require a light period to induce sporulation. In this paper the ultrastructure and acid phosphatase localization of the mature plasmodium ofPerichaena vermicularis are investigated. Acid phosphatase is localized in vacuoles containing remnants of bacteria and cell organelles. Morphological and histochemical evidence support the interpretation that these vacuoles constitute two types of lysosomes called respectively heterophagic and autophagic vacuoles.Coated vesicles which apparently originate from smooth endoplasmic reticulum are dispersed throughout the plasmodium and frequently associated with lysosomes. Several dumbbellshaped mitochondria are observed in the plasmodium at the onset of fruiting but not during later stages of plasmodiocarp development. Cytoplasmic microtubules are identified inPerichaena vermicularis. Some of these are closely associated with microfilaments.

Syngamy and plasmodium formation in the MyxomyceteDidymium iridis

SummaryCell and nuclear behavior during syngamy in the MyxomyceteDidymium iridis and the development of zygotes into plasmodia by both synchronous mitoses and by coalescence with other zygotes and plasmodia are described. Various aspects of cell and nuclear behavior are discussed in relation to the induction of syngamy and the trigger mechanisms responsible for switching the course of development from one pathway to another.

DYNAMIC ASPECTS OF BASIDIOSPORE NUMBER IN AGARICUS

The average number of basidiospores produced upon the basidium of the nominally tetrasporic Agaricus subrufescens can vary considerably. In the laboratory a decline in average spore number can be induced by refrigerating the sporocarps for several hours. Spore number also fluctuates in naturally occurring material, in a manner which correlates with diunal temperature fluctuations. Observations on several other species of Agaricus suggest that this phenomenon is widespread in the genus. These findings raise questions about the life cycle of these organisms, and suggest an explanation for the origin of bisporic, secondarily homothallic species such as A. bisporus.

Cloning and characterization of a G protein α-subunit-encoding gene from the basidiomycete, Coprinus congregatus

Complementary DNA (cDNA) clones encoding two G protein alpha-subunit proteins (CGP alpha 1 and CGP alpha 2) were isolated from a Coprinus congregatus (Cc) hyphal tip cell (HTC) library using PCR-generated biotinylated G protein probes. Sequence analysis of the Cc cgp alpha 1 gene indicates that the gene contains an open reading frame (ORF) that translates into a putative 353-amino-acid (aa) product. The predicted CGP alpha 1 protein exhibits similarity to all known G protein alpha-subunits (it has all of the consensus regions for a GTP-binding protein), especially the mammalian retinal G protein, transducin. The CGP alpha 1 aa sequence is 50% identical overall to the transducin subfamily, cgp alpha 1 shares the same aa size grouping as transducin alpha-subunits and, unlike many other G proteins, both CGP alpha 1 and transducin seem to possess a cholera toxin (CTX)- and pertussis toxin (PTX)-sensitive site. Preliminary reverse transcription PCR (RT-PCR) analysis of cgp alpha 1 and cgp alpha 2 mRNA expression revealed that, unlike cgp alpha 2 which seems to be constitutively expressed, cgp alpha 1 is expressed only in HTC that are competent in responding to light. Thus, the cgp alpha 1 product, CGP alpha 1, is a likely candidate for regulating the blue light-induced signal transduction photomorphogenesis system found in Cc.

Nuclear membrane behavior during mitosis in normal and heteroploid myxomycetes.

SummaryPhotomicrographic evidence is presented of the difference in behavior of nuclear membranes during mitosis in amoebae, zygotes and plasmodia of Myxomycetes. One of the species was cultured on bacteria and possessed a normal cycle of plasmogamy and karyogamy between the amoebal and plasmodial phases. The second species was axenically grown in liquid media and had become highly heteroploid and lacked the ability to develop into plasmodia, existing only in the amoeboid form. The significance of the amoeboid form of mitosis in the heteroploid axenically cultured strain is discussed in relation to the difference in nuclear membrane behavior and the possible significance of such behavior.