Shawn K. Stickel

Evolutionary relationships within the fungi: Analyses of nuclear small subunit rRNA sequences

Nucleotide sequences of the small subunit ribosomal RNA (18S) gene were used to investigate evolutionary relationships within the Fungi. The inferred tree topologies are in general agreement with traditional classifications in the following ways: (1) the Chytridiomycota and Zygomycota appear to be basal groups within the Fungi. (2) The Ascomycota and Basidiomycota are a derived monophyletic group. (3) Relationships within the Ascomycota are concordant with traditional orders and divide the hemi- and euascomycetes into distinct lineages. (4) The Basidiomycota is divided between the holobasidiomycetes and phragmobasidiomycetes. Conflicts with traditional classification were limited to weakly supported branches of the tree. Strongly supported relationships were robust to minor changes in alignment, method of analysis, and various weighting schemes. Weighting, either of transversions or by site, did not convincingly improve the status of poorly supported portions of the tree. The rate of variation at particular sites does not appear to be independent of lineage, suggesting that covariation of sites may be an important phenomenon in these genes.

Morphological and genetic variation within the diatom Skeletonema costatum (Bacillariophyta) : evidence for a new species, Skeletonema pseudocostatum

The nuclear gene encoding the small subunit (16S‐like) ribosomal RNA molecule was sequenced from four isolates of the marine coastal planktonic diatom Skeletonema costatum (Grev.) Cleve. Each isolate originated from a geographically separate water mass. Sufficient morphological and genetic differences were found among the four isolates to support the description of a new species, Skeletonema pseudocostatum, which corresponds to the CCAP 1077/7 and CS‐76 isolates.

Phylogeny of trichomonads inferred from small-subunit rRNA sequences.

ABSTRACT. Small subunit (16S‐like) ribosomal RNA sequences were obtained from representatives of all four families constituting the order Trichomonadida. Comparative sequence analysis revealed that the Trichomonadida are a monophyletic lineage and a deep branch of the eukaryotic tree. Relative to other early divergent eukaryotic assemblages the branching pattern within the Trichomonadida is very shallow. This pattern suggests the Trichomonadida radiated recently, perhaps in conjunction with their animal hosts. From a morphological perspective the Devescovinidae and Calonymphidae are considered more derived than the Monocercomonadidae and Trichomonadidae. Molecular trees inferred by distance, parsimony and likelihood techniques consistently show the Devescovinidae and Calonymphidae are the earliest diverging lineages within the Trichomonadida, however bootstrap values do not strongly support a particular branching order. In an analysis of all known 16S‐like ribosomal RNA sequences, the Trichomonadida share most recent common ancestry with unidentified protists from the hindgut of the termite Reticulitermes flavipes. The position of two putative free‐living trichomonads in the tree is indicative of derivation from symbionts rather than direct descent from some free‐living ancestral trichomonad.

Algae containing chlorophylls a + c are paraphyletic: molecular evolutionary analysis of the Chromophyta

Sequence comparisons of small subunit ribosomal RNA coding regions from 12 chlorophylls a + c‐containing algae were used to infer phylogenetic relationships within the Chromophyta. Three chromophyte lines of descent, delineated by the Bacillariophyceae, the Phaeophyceae/Xanthophyceae, and the Chrysophyceae/Eustigmatophyceae/Synurophyceae are members of a complex evolutionary assemblage, which also includes representatives of the Oomycota (“lower” fungi). Maximum parsimony and distance matrix methods demonstrate a common evolutionary history for these lineages but their relative branching order could not be determined. Other algal species with chlorophylls a + c, including dinoflagellates and prymnesiophytes, are not members of this complex assemblage. Dinoflagellates are specifically related to apicomplexans and ciliates, and the prymnesiophyte, Emiliania huxleyi, represents an independent photosynthetic lineage that separated from other eukaryotes during the nearly simultaneous divergence of plants, animals, fungi, and a number of other protist lineages. The small subunit rRNA phylogenies of chromophytes/oomycetes were compared to those derived from comparisons of ultrastructural characters. Only tubular, tripartite mastigonemes (flagellar hairs) characterized all studied taxa of chromophytes/oomycetes as a monophyletic assemblage.

Molecular phylogenetic analysis of actin genic regions from Achlya bisexualis (Oomycota) and Costaria costata (Chromophyta).

SummaryActin genic regions were isolated and characterized from the heterokont-flagellated protists,Achlya bisexualis (Oomycota) andCostaria costata (Chromophyta). Restriction enzyme and cloning experiments suggested that the genes are present in a single copy and sequence determinations revealed the existence of two introns in theC. costata actin genic region. Phylogenetic analyses of actin genic regions using distance matrix and maximum parsimony methods confirmed the close evolutionary relationship ofA. bisexualis andC. costata suggested by ribosomal DNA (rDNA) sequence comparisons and reproductive cell ultrastructure. The higher fungi, green plants, and animals were seen as monophyletic groups; however, a precise order of branching for these assemblages could not be determined. Phylogenetic frameworks inferred from comparisons of rRNAs were used to assess rates of evolution in actin genic regions of diverse eukaryotes. Actin genic regions had nonuniform rates of nucleotide substitution in different lineages. Comparison of rates of actin and rDNA sequence divergence indicated that actin genic regions evolve 2.0 and 5.3 times faster in higher fungi and flowering plants, respectively, than their rDNA sequences. Conversely, animal actins evolve at approximately one-fifth the rate of their rDNA sequences.

REGULATION OF SCD4-183 GENE EXPRESSION FROM PHAGE-T7-BASED VECTORS IN ESCHERICHIA COLI

In this paper, we describe various parameters affecting the regulation of expression of the sCD4-183 gene, encoding the 183-amino-acid soluble human two-domain CD4 protein, from phage-T7-based pET vectors. We demonstrated that for the sCD4-183 protein, the highest protein yield was obtained using vector pET-9a, in which neither expression of the T7 RNA polymerase-encoding gene nor the target gene was tightly regulated. The highest overall protein yield was obtained from cells grown for 24 h in the absence of inducer, a strategy that may be generally useful for production of less toxic proteins. We also describe two modifications of the pET vector system that effectively minimized leaky (uninduced) expression and enhanced plasmid stability. These have potential use in the production of toxic proteins, or of non-toxic proteins produced in high-density cultures.

Sequence analysis of duplicated actin genes in Lagenidium giganteum and Pythium irregulare (Oomycota)

Southern analysis of genomic DNA identified multiple-copy actin gene families in Lagenidium giganteum and Pythium irregulare (Oomycota). Polymerase chain reaction (PCR) protocols were used to amplify members of these actin gene families. Sequence analysis of genomic coding regions demonstrated five unique actin sequences in L. giganteum (Lg-Ac 1, 2, 3, 4, 5) and four unique actin sequences in P. irregulare (Pi-Acl, 2, 3, 4); none were interrupted by introns. Maximum parsimony analysis of the coding regions demonstrated a close phylogenetic relationship between oomycetes and the chromophyte alga Costaria costata. Three types of actin coding regions were identified in the chromophyte/oomycete lineage. The type 1 actin is the single-copy coding region found in C. costata. The type 2 and type 3 actins are found in the oomycetes and are the result of a gene duplication which occurred soon after the divergence of the oomycetes from the chromophyte algae. The type 2 coding regions are the single-copy sequence of Phytophthora megasperma, the Phytophthora infestans actB gene, Lg-Ac5 and Pi-Ac2. The type 3 coding regions are the single-copy sequence of Achlya bisexualis, the P. infestans actA gene, Lg-Ac1, 2, 3, 4 and Pi-Acl, 3, 4.