Ron M. MacKay

REVEALING GENETIC MARKERS IN GELIDIUM VAGUM (RHODOPHYTA) THROUGH THE RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) TECHNIQUE1

The recently developed random amplified polymorphic DNA technique was evaluated as a method for characterizing isolates of the agarophyte Gelidium vagum Okamura. Reaction conditions for single primer polymerase chain reaction were optimized to obtain a high degree of reproducibility of the amplified bands generated from purified G. vagum DNA. A total of 165 primers, including both (A + T)‐ and (G + C)‐rich sequences, was screened for DNA amplification using template DNA from a single Gelidium isolate. None of the 45 (A + T)‐rich primers was positive (i.e. band‐producing). Of the (G + C)‐rich primers, 47 were positive, generating a total of 322 prominent amplification products for DNA from 13 different G. vagum isolates. Polymorphic DNA loci were detected by 37 of the primers. Unweighted pair‐group arithmetic average cluster analysis (UPGMA) of these loci was used to group the G. vagum isolates and thereby determine which were most similar. G. latifolium, used as an out‐group for the UPGMA analysis, showed a high degree of dissimilarity.

Molecular cloning of three cDNAs that encode cysteine proteinases in the digestive gland of the American lobster (Homarus americanus)

Abstract Three clones were isolated from a lobster digestive gland cDNA library, using oligonucleotide probes based on the partial amino terminal sequence of a digestive cysteine proteinase. The cDNAs, LCP1, LCP2 and LCP3 encode preproenzymes of 322, 323 and 321 amino acid residues, and putative mature enzymes of 217, 216 and 215 residues, respectively. Calculated mature protein molecular masses are 23386 (LCP1), 23093 (LCP2) and 23255 (LCP3) Sequence alignments show that the lobster enzymes are more similar to L (55–62% identity) than H (42–44%) or B (22–24%) cathepsins. Southern analysis indicated as many as eleven genes related to the three cDNAs.Southern analysis indicated as many as eleven genes related to the three cDNAs.

The origin of plant chloroplast 4.5 S ribosomal RNA

Nazar [l] has recently pointed out the extensive homology (-53%) between trout 5.8 S rRNA (ribosomal ribonucleic acid) and the 5’-terminus of Escherichia coli 23 S rRNA, and has suggested that the prokaryotic homologue of eukaryotic 5.8 S exists not as an independent RNA species, but as the 5’-terminus of the 23 S rRNA. This concept is consistent with the observation that the eukaryotic 5.8 S sequence is located proximal to the 5’-end of the 28 S rRNA sequence in a larger precursor RNA species [ 11. This approach is used here to consider another small rRNA, the 4.5 S rRNA found in some chloroplast ribosomes [2]. The 4.5 S gene is located 3’ to the 23 S gene and 5’ to the 5 S gene in tobacco chloroplast DNA [3]. This suggests that chloroplast 4.5 S rRNA, with no other known homologue in other types of ribosomes, may be homologous to the 3’-terminus of prokaryotic 23 S rRNA. The complete sequence of E. coli 23 S rRNA [4] and the recently reported sequence of tobacco chloroplast 4.5 S rRNA [5] allows this hypothesis to be tested. A HO GAAGilJCA6iGCG~~ACGA2GOCCGUt;UAC

Discrimination between Hirsutella longicolla var. longicolla and hirsutella longicolla var. cornuta using random amplified polymorphic DNA fingerprinting

The synnematous, fungal entomopathogen Hirsutella longicolla var. longicolla is distinguished from a variety Hirsutella longicolla var. cornuta on the basis of the synnematal morphology. Random Amplified Polymorphic DNA (RAPD) fingerprinting supports the view that these fungi are genetically distinct. Two different banding patterns, one corresponding to isolates of the species and the other to the variety, were generated with two primers and DNA from 19 isolates. Further analysis using nine isolates (four of the species and five of the variety) with three additional primers confirmed the distinction between the species and its variety. This work illustrates the usefulness of the Random Amplified Polymorphic DNA technique in addressing taxonomic questions.

Glucanase gene diversity in prokaryotic and eukaryotic organisms.

A number of bacteria and eukaryotes produce extracellular enzymes that degrade various types of polysaccharides including the glucans starch, cellulose and hemicellulose (xylan). The similarities in the modes of expression and specificity of enzyme classes, such as amylase, cellulose and xylanase, suggest common genetic origins for particular activities. Our determination of the extent of similarity between these glucanases suggests that such data may be of very limited use in describing the early evolution of these proteins. The great diversity of these proteins does allow identification of their most highly conserved (and presumably functionally important) regions.

NUCLEOTIDE SEQUENCES OF SMALL-SUBUNIT AND INTERNAL TRANSCRIBED SPACER REGIONS OF NUCLEAR rRNA GENES SUPPORT THE AUTONOMY OF SOME GENERA OF THE GELIDIALES (RHODOPHYTA)

The lack of homogeneity in all previously proposed, distinguishing characteristics has left the relationships of taxa within the Gelidiales as one of the most enduring taxonomic uncertainties in the Rhodophyta. Although a precise knowledge of the taxonomy of commercially harvested members of the Gelidiales would assist resource management, agronomic practices, and marketing, even the distinction between two major groups, Gelidium and Pterocladia, has long remained controversial. In this study, the 18S ribosomal RNA (rRNA), internal transcribed spacer 1 (ITS1), 5.8S rRNA, and ITS2 regions of Gelidium latifolium (Greville) Bornet et Thuret, G. sesquipedale (Clemente) Thuret in Bornet et Thuret, G. vagum Okamura, Pterocladia lucida (Brown ex Turner) J. Agardh, and a recent segregate from Pterocladia, Pterocladiella capillacea (Gmelin) Santelices et Hommersand, were sequenced and analyzed. The ITS1, 5.8S rRNA, and ITS2 regions of G. arbuscula (Montagne) B=orgesen, G. canariensis (Grunow) Seoane‐Camba, G. capense (Gmelin) Silva, and G. pristoides (Turner) Kützing were also sequenced. Phylogenetic analyses based on the 18S rRNA genes from four Gelidium species, Pterocladia lucida, and Pterocladiella capillacea, compared with 18S rRNA genes from several other red algae confirmed the division between Gelidium and the Pterocladia/Pterocladiella isolates and were consistent with the recently proposed segregation of Pterocladiella from Pterocladia. Analyses based on the ITS regions of seven Gelidium species, Pterocladia lucida, and Pterocladiella capillacea were completely consistent with the conclusions drawn from the 18S rRNA data. There were extensive length and sequence differences between Gelidium and Pterocladia/Pterocladiella. In addition, there were larger sequence differences between Pterocladiella and Pterocladia than exist among Gelidium isolates, in keeping with the recently proposed separation of the former two taxa.

Comparisons of twenty isolates of the entomopathogen Paecilomyces farinosus by analysis of RAPD markers

The genetic relatedness of twenty isolates of Paecilomyces farinosus was determined by comparison of the products of polymerase chain reaction amplification of anonymous regions of genomic DNA with single arbitrary sequence oligonucleotide primers (RAPD analysis). Isolates were collected from seven insect species in eastern Canada and they differed greatly in cultural and morphological phenotype. All P. farinosus isolates were clearly distinguished from three other entomopathogenic fungi, including P. fumosoroseus. RAPD banding patterns did not, however, correlate with ecological backgrounds or morphological phenotypes of P. farinosus isolates. These observations support the conclusion that P. farinosus from eastern Canada is not composed of strains which can be separated on the basis of the ecological or morphological criteria selected.

Beta-tubulins are encoded by at least four genes in the brown algaEctocarpus variabilis

Complementary DNA clones of two mRNA species that encode β-tubulin in the brown algaEctocarpus variabilis have been isolated. Sequence analysis revealed that the encoded proteins are very similar in primary structure to homologues in other eukaryotes, and differ from each other at six of 447 amino acid residues. The β6 message shows a preference for C-or G-terminated codons, using only 49 codons. The β5 message has a lesser codon bias, and makes a minor contribution to the β-tubulin mRNA pool. Southern analysis ofE. variabilis DNA demonstrated a β-tubulin gene family of at least four members.

β‐TUBULIN GENE OF PORPHYRA PURPUREA ( RHODOPHYTA)

The life cycle of the marine red alga Porphyra purpurea (Roth) C. Agardh includes a shell‐boring filamentous sporophyte and a leafy gametophyte. A single intronless gene for the microtubule protein β‐tubulin was discovered by molecular cloning of P. purpurea cDNA and genomic DNA. This gene, named TubB1, encodes a β‐tubulin with a divergent amino acid sequence, showing 74% identity with the conserved β‐tubulin of Chlamydomonas reinhardtii P. A. Dangeard. Southern hybridization analysis of nuclear DNA confirmed that P. purpurea has a single TubB1 gene. Transcripts (1.8 kb) of TubB1 are present in the sporophyte and gametophyte. Codon bias indicates strong expression of TubB1. The divergent nature of the TubB1 genes suggests that the absence of axonemal structures has allowed substantial genetic drift in red algal β‐tubulin genes.