Benjamin D. Hall

Sequence of the gene for iso-l-cytochrome c in saccharomyces cerevisiae

Abstract The complete sequence of the iso-1-cytochrome c gene of yeast has been determined. The coding region of the gene contains no intervening sequences. The coding strand of the DNA immediately upstream from the coding region contains many fewer G residues than the rest of the coding strand both within and beyond the carboxy terminus of the coding region. One consequence of the reduced number of G residues in this region is the absence of the sequence ATG for 122 nucleotides upstream from the initiating ATG. Together with previous studies on the mRNA and the genetics of yeast iso-1-Cytochrome c, the sequence supports a model in which translation starts at the first AUG down-stream from the 5′ terminus of the mRNA, with no other sequence requirements. It also is evident that iso-1-cytochrome c is synthesized directly and not through an intermediary, longer precursor protein, as is often the case for proteins that interact with membranes. The DNA upstream and downstream from the coding region contains sequences which are potential transcription start and stop signals. The sequence confirms the assignments of non-sense and missense mutations throughout the coding region of the gene and provides a rationale for some mutational characteristics of the gene. Part of the sequence was determined using two new strategies for the Sanger terminator method, both of which obviate the need for restriction fragment isolation and template strand separation.

Loss of the flagellum happened only once in the fungal lineage: phylogenetic structure of Kingdom Fungi inferred from RNA polymerase II subunit genes

BackgroundAt present, there is not a widely accepted consensus view regarding the phylogenetic structure of kingdom Fungi although two major phyla, Ascomycota and Basidiomycota, are clearly delineated. Regarding the lower fungi, Zygomycota and Chytridiomycota, a variety of proposals have been advanced. Microsporidia may or may not be fungi; the Glomales (vesicular-arbuscular mycorrhizal fungi) may or may not constitute a fifth fungal phylum, and the loss of the flagellum may have occurred either once or multiple times during fungal evolution. All of these issues are capable of being resolved by a molecular phylogenetic analysis which achieves strong statistical support for major branches. To date, no fungal phylogeny based upon molecular characters has satisfied this criterion.ResultsUsing the translated amino acid sequences of the RPB1 and RPB2 genes, we have inferred a fungal phylogeny that consists largely of well-supported monophyletic phyla. Our major results, each with significant statistical support, are: (1) Microsporidia are sister to kingdom Fungi and are not members of Zygomycota; that is, Microsporidia and fungi originated from a common ancestor. (2) Chytridiomycota, the only fungal phylum having a developmental stage with a flagellum, is paraphyletic and is the basal lineage. (3) Zygomycota is monophyletic based upon sampling of Trichomycetes, Zygomycetes, and Glomales. (4) Zygomycota, Basidiomycota, and Ascomycota form a monophyletic group separate from Chytridiomycota. (5) Basidiomycota and Ascomycota are monophyletic sister groups.ConclusionIn general, this paper highlights the evolutionary position and significance of the lower fungi (Zygomycota and Chytridiomycota). Our results suggest that loss of the flagellum happened only once during early stages of fungal evolution; consequently, the majority of fungi, unlike plants and animals, are nonflagellated. The phylogeny we infer from gene sequences is the first one that is congruent with the widely accepted morphology-based classification of Fungi. We find that, contrary to what has been published elsewhere, the four morphologically defined phyla (Ascomycota, Basidiomycota, Zygomycota and Chytridiomycota) do not overlap with one another. Microsporidia are not included within kingdom Fungi; rather they are a sister-group to the Fungi. Our study demonstrates the applicability of protein sequences from large, slowly-evolving genes to the derivation of well-resolved and highly supported phylogenies across long evolutionary distances.

Identification and isolation of the yeast cytochrome c gene

The iso-1-cytochrome c gene of yeast has been identified and cloned using a synthetic oligodeoxynucleotide as a hybridization probe. The oligomer d[pT-T-A-G-C-A-G-A-A--C-C-G-G] is complementary to a region near the N terminal coding region of the yeast cyc 1 gene. Of several yeast Eco RI fragments which hybridize to this probe, one is changed in size by a G leads to T mutation which eliminates an Eco RI site within the cyc 1 gene. Both the wild-type and the RI- mutant forms were cloned in lambda gt vectors. Maxam-Gilbert sequencing for 91 nucleotides into the coding region for iso-1-cytochrome c yielded a DNA sequence in perfect correspondence with the known protein sequence.

Mutations of the yeast SUP4 tRNATyr Locus: Transcription of the mutant genes in vitro

Twenty-nine different SUP4-o tRNATyr genes with second-site mutations were transcribed in X. laevis cell-free RNA polymerase III transcription reactions, and the in vitro transcripts were analyzed by polyacrylamide gel electrophoresis. Nineteen mutant genes yield normal amounts of RNA that co-electrophorese with SUP4-o gene transcripts. RNA synthesized from a mutant gene lacing a single base pair migrated slightly faster in gels, as expected. The still shorter transcripts made from seven other mutant genes suggest that several mutations alter transcription starting or stopping points. Fingerprint analyses of transcripts from the two most extreme cases showed that premature termination occurred at new tracts of T residues resulting from the mutations. Two mutations significantly enhance transcription, and two mutations which alter the invariant C within the T psi CG sequence dramatically reduce SUP4-o gene transcription. The regions of the SUP4-o gene that surround these mutations are partially homologous to intragenic sequences in many other eucaryotic tRNA and 5S RNA genes. We hypothesize that these homologous sequences are recognized as promoter regions during RNA polymerase III transcription initiation.

Evolution of the RNA polymerase II C-terminal domain

In recent years a great deal of biochemical and genetic research has focused on the C-terminal domain (CTD) of the largest subunit (RPB1) of DNA-dependent RNA polymerase II. This strongly conserved domain of tandemly repeated heptapeptides has been linked functionally to important steps in the initiation and processing of mRNA transcripts in both animals and fungi. Although they are absolutely required for viability in these organisms, C-terminal tandem repeats do not occur in RPB1 sequences from diverse eukaryotic taxa. Here we present phylogenetic analyses of RPB1 sequences showing that canonical CTD heptads are strongly conserved in only a subset of eukaryotic groups, all apparently descended from a single common ancestor. Moreover, eukaryotic groups in which the most complex patterns of ontogenetic development occur are descended from this CTD-containing ancestor. Consistent with the results of genetic and biochemical investigations of CTD function, these analyses suggest that the enhanced control over RNA polymerase II transcription conveyed by acquired CTD/protein interactions was an important step in the evolution of intricate patterns of gene expression that are a hallmark of large, developmentally complex eukaryotic organisms.

The Molecular Systematics of Rhododendron (Ericaceae): A Phylogeny Based Upon RPB2 Gene Sequences

Abstract Classification of Rhododendron species based on morphology has led to a consensus taxonomy recognizing the major subgenera Azaleastrum, Hymenanthes, Pentanthera, Rhododendron, Tsutsusi, and three minor ones. To determine whether these subgenera are monophyletic and to infer phylogenetic relationships between Rhododendron sections and species, we carried out a cladistic analysis using molecular data, including all groups within the genus. For this purpose, we sequenced a large part of the nuclear gene RPB2-I, encoding a major RNA Polymerase II subunit, from 87 species and analyzed the data by maximum parsimony, maximum likelihood, and Bayesian methods. The resulting phylogenies show subgenera Azaleastrum and Pentanthera to be polyphyletic and group all Rhododendron species (except the two in section Therorhodion) into three large clades. Based upon these results, modifications in Rhododendron classification are proposed, which consolidate minor subgenera and recognize monophyletic subgenera and sections.

Are Red Algae Plants? A Critical Evaluation of Three Key Molecular Data Sets

Abstract. Whether red algae are related to green plants has been debated for over a century. Features present due to their shared photosynthetic habit have been interpreted as support for an evolutionary sisterhood of the two groups but, until very recently, characters endogenous to the host cell have provided no reliable indication of such a relationship. In this investigation, we examine three molecular data sets that have provided key evidence of a possible relationship between green plants and red algae. Analyses of an expanded alignment of DNA-dependent RNA polymerase II largest subunit sequences indicate that their support for independent origins of rhodophytes and chlorophytes is not the result of long-branch attraction, as has been proposed elsewhere. Differences in the pol II C-terminal domain, an essential component of plant mRNA transcription, also suggest different host cell ancestors for the two groups. In contrast, concatenated sequences of two groups of mitochondrial genes, those encoding subunits of NADH-dehydrogenase as well as cytochrome c oxidase subunits plus apocytochrome B, appear to cluster red algal and green plant sequences together because both groups have evolved relatively slowly and share a super-abundance of ancestral positions. Finally, analyses of elongation factor 2 sequences demonstrate a strong phylogenetic signal favoring a rhodophyte/chlorophyte sister relationship, but that signal is restricted to a contiguous segment comprising approximately half of the EF2 gene. These results argue for great caution in the interpretation of phylogenetic analyses of ancient evolutionary events but, in combination, indicate that there is no emerging consensus from molecular data supporting a sister relationship between red algae and green plants.

Transcription initiation of eucaryotic transfer RNA Genes

RNA polymerase Ill (Pal Ill), an enzyme found in the nuclei of animals, plants and fungi, has been implicated in the in vivo transcription of 5s rRNA, pretRNA, some small viral RNAs and the cellular RNAs derived from certain middle-repetitive genomic sequences. It is a complex -700 kd protein composed of at least ten distinct subunits. The enzyme is not able to transcribe purified genes with fidelity by itself but requires additional components whose number, nature and modes of action are only just beginning to be characterized. Much more is known about the locations of the DNA signals that permit accurate transcription initiation of some Pol Ill genes. This information has come primarily from a two-step experimental approach. In the first step, DNA is progressively deleted from around and within the gene in question; the deleted sequences are then replaced by heterologous DNA. Alternatively, single-base changes or short deletions are introduced into the gene by in vivo or in vitro mutagenesis. The second step is then to assay the transcriptional effects of these sequence manipulations by microinjection of the mutant genes into the nuclei of frog oocytes or by their incubation in a variety of in vitro transcription systems. We shall compare and contrast the results of such analyses with several Pol Ill genes and discuss, in particular, the structure and function of the DNA signals important for tRNA gene transcription initiation. Pol III Promoters Are lntragenic Deletion analyses have shown that the only DNA sequences essential for transcription of a frog 5s RNA gene are located between residues 50 and 83 (Sakonju et al., Cell 79, 13-25, 1980; Bogenhagen et al., Cell 79, 27-35, 1980). A plausible basis for the mode of action of this intragenic promoter was provided by Engelke et al. (Cell 79, 717-728, 19801, who showed that the same region of the 5s RNA gene binds to a 5S-specific transcription factor. These results suggest that this 37 kd transcription factor, once bound, may interact with a Pol III molecule to position its catalytic sites on the transcription start point of the DNA. The same resection approach has shown that tRNA genes also contain intragenic promoters. Their maximum boundaries have been defined as residues 8 and 62 within the genes encoding the initiator tRNAMe’ (Hofstetter et al., Cell 24, 573-585, 1981) and tRNA% (Galli et al., Nature 294, 626-631, 1981) of X. laevis and the tRNAP”’ of Caenorhabditis elegans (Ciliberto et al., PNAS 79, 1195-l 199, 1982). Unlike their counterparts within 5s genes, however, the essential nucleotides are split into two sequence blocks that are set far apart. These sequences, termed the A and B blocks (Galli et al., op. cit.), have the approximate coordinates 8-19 and 52-62, respectively, by the standard system of numbering tRNA genes. Their locations with respect to the tRNA cloverleaf are indicated in Figure 1. Two lines of evidence in particular support the notion of discontinuous intragenic promoters: chimeric tRNA genes containing the 5’ half of one gene and the 3’ half of another can be transcribed well; and transcription can also occur after the replacement of the central region of tRNA genes with DNA of very different sequence (Ciliberto et al., op. cit.; Galli et al., op. cit.). These central regions do appear to have a spacing function, however, because the efficiency of transcription depends on the length of the replaced DNA, the optimal distance between the A and B blocks being 30-40 bp (Ciliberto et al., PNAS 79, 19211925, 1982). In natural tRNA genes this distance can vary from 31 to >74 bp, the variability being due to the length of the V arm and the presence within certain tRNA genes of an intervening sequence. Although not absolutely required for transcription (Wallace et al., Science 209, 1396-1400, 1980), intervening sequences may still influence the efficiency of this process by expanding the distance between the A and B blocks, thereby diminishing promoter strength. An unusual feature of tRNA gene promoters is thus the great latitude in distance between the A and B blocks. In contrast, the distance between the transcription start point and the A block is less variable (10-I 6 bp). Another intriguing feature of these sequences is their close correlation with the most con-

Identification of the yeast DNA sequences that correspond to specific tyrosine-inserting nonsense suppressor loci☆

Abstract There are eight unlinked genes for yeast tyrosine transfer RNA. In previous work, nonsense suppressors have been isolated at each of the eight loci, and these loci have been genetically mapped (Hawthorne & Leupold, 1974). It has also been demonstrated by RNA-DNA hybridization that the genes are physically located on eight different Eco RI restriction fragments (Olson et al. , 1977). The purpose of the present report is to cross-correlate the set of tyrosine-inserting suppressor loci with the set of tRNA Tyr -hybridizing restriction fragments. This cross-correlation was achieved for six of the eight loci by analyzing the meiotic and mitotic linkage between the tyrosine-inserting suppressors and the genetic determinants of naturally occurring size variants of the tRNA Tyr -hybridizing restriction fragments. Now that individual suppressor loci have been identified with specific DNA fragments, it should be possible to analyze the phenotypes of these mutant genes in terms of their DNA sequences. The method by which these assignments were made also offers a new approach to the general problem of correlating genes with restriction fragments; it is particularly suited to organisms with powerful genetic systems in which hybridization to chromosome spreads in situ is impractical.

A multistep process gave rise to RNA polymerase IV of land plants.

Since their discovery in Metazoa, the three nuclear RNA polymerases (RNAPs) have been found in fungi, plants, and diverse protists. In all eukaryotes studied to date, RNAPs I, II, and III collectively transcribe all major RNAs made in the nucleus. We have found genes for the largest subunit (RPD1/RPE1) of a new DNA-dependent RNAP, RNAP IV, in all major land plant taxa and in closely related green algae. Genes for the second-largest subunit (RPD2) of this enzyme were found in all land plants. Phylogenetic study indicates that RNAP IV genes are sister to the corresponding RNAP II genes. Our results show the genesis of RNAP IV to be a multistep process in which the largest and second-largest subunit genes evolved by independent duplication events in the ancestors of Charales and land plants. These findings provide insights into evolutionary mechanisms that can explain the origin of multiple RNAPs in the eukaryotic nucleus.