Biological speciation ultimately results in prezygotic isolation-the inability of incipient species to mate with one another-but little is understood about the selection pressures and genetic changes that generate this outcome. The genus Chlamydomonas comprises numerous species of unicellular green algae, including numerous geographic isolates of the species C. reinhardtii. This diverse collection has allowed us to analyze the evolution of two sex-related genes: the mid gene of C. reinhardtii, which determines whether a gamete is mating-type plus or minus, and the fus1 gene, which dictates a cell surface glycoprotein utilized by C. reinhardtii plus gametes to recognize minus gametes. Low stringency Southern analyses failed to detect any fus1 homologs in other Chlamydomonas species and detected only one mid homolog, documenting that both genes have diverged extensively during the evolution of the lineage. The one mid homolog was found in C. incerta, the species in culture that is most closely related to C. reinhardtii. Its mid gene carries numerous nonsynonymous and synonymous codon changes compared with the C. reinhardtii mid gene. In contrast, very high sequence conservation of both the mid and fus1 sequences is found in natural isolates of C. reinhardtii, indicating that the genes are not free to drift within a species but do diverge dramatically between species. Striking divergence of sex determination and mate recognition genes also has been encountered in a number of other eukaryotic phyla, suggesting that unique, and as yet unidentified, selection pressures act on these classes of genes during the speciation process.Sexual eukaryotes carry two classes of sex-related genes: sex determination genes act in individual organisms to determine their gender or mating type, and mate recognition genes encode traits that assure that mating occurs between the correct gender͞mating type of the correct species. We have cloned and characterized a gene of each class in the unicellular green alga Chlamydomonas reinhardtii. The sex determination mid (minus dominance) gene encodes a regulatory protein that is necessary to express the mating type minus sexual differentiation program and to switch off the mating type plus program (1). The mate recognition fus1 gene encodes a cell surface glycoprotein called fringe that is necessary for plus gametes to adhere to minus gametes and subsequently to fuse to form zygotes (2). The mid gene is located in a highly rearranged region, the R domain, of the mating-type minus (mt Ϫ ) locus and is unique to the mt Ϫ chromosome; the fus1 gene is located in the R domain of the mt ϩ locus and is unique to the mt ϩ chromosome (3). This study reports the results of experiments designed to identify, by low stringency hybridization, mid and fus1 homologs in other members of the Volvocales, focusing in particular on species that have been found, by cladistic analysis (4), to be near relatives of C. reinhardtii. No fus1 homologs were detected, and only one mid homolog was detected, indicating...

The glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei (optimal growth temperature, 100 to 103°C) was purified to homogeneity. This enzyme was strictly phosphate dependent, utilized either NAD+ or NADP+, and was insensitive to pentalenolactone like the enzyme from the methanogenic archaebacterium Methanothermus fervidus. The enzyme exhibited a considerable thermostability, with a 44-min half-life at 100°C. The amino acid sequence of the glyceraldehyde-3-phosphate dehydrogenase from P. woesei was deduced from the nucleotide sequence of the coding gene. Compared with the enzyme homologs from mesophilic archaebacteria (Methanobacterium bryantii, Methanobacterium formicicum) and an extremely thermophilic archaebacterium (Methanothermusfervidus), the primary structure of the P. woesei enzyme exhibited a strikingly high proportion of aromatic amino acid residues and a low proportion of sulfur-containing residues. The coding gene ofP. woesei was expressed at a high level in Escherichia coli, thus providing an ideal basis for detailed structural and functional studies of that enzyme.The extraordinary ability to grow above 100°C is exclusively restricted to certain members of archaebacteria. These hyperthermophilic strains with optimal growth temperatures ranging from 100 to 105°C are represented by four genera: Pyrodictium (41), Pyrococcus (19), Pyrobaculum (26), and Methanopyrus (27).Although the hyperthermophily brings up evident questions concerning the molecular background of thermoadaptation, knowledge of the macromolecular cell constituents of these organisms is scarce. Thus, with the exception of the reports on hydrogenase and ferredoxin from Pyrococcus furiosus (5, 10), no description of any protein from these hyperthermophiles has yet been published.Here we characterize some phenotypic properties of the glyceraldehyde-3-phosphate dehydrogenase from Pyrococcus woesei (optimal growth temperature, 100 to 103°C [47]) and report on the cloning and sequencing of the coding gene as well as on its expression in Escherichia coli.To get indications about the structural adaptation to the extreme growth temperatures, we compared the sequence of the glyceraldehyde-3-phosphate dehydrogenase of P. woesei with the structures of the enzyme homologs from mesophilic and thermophilic archaebacteria (17). MATERIALS AND METHODSBacterial strains. Cells of P. woesei Vul4 (DSM 3773) were grown as described previously (47). For cloning and expression of the glyceraldehyde-3-phosphate dehydrogenase gene, the E. coli K-12 strains JM83 [ara A(lac-proAB) strA thi 4)80dlacZAM15 (33)] and DH5a [F-endAl hsdRJ7 (rK-MK+) supE44 thi-J X-recAl gyrA96 relAl A(lacZYAargF)U169 4.80dlacZAM15 (28)] were used.Plasmids, enzymes, chemicals. The vectors for cloning and sequencing were pUC18 and M13mpl8/19 (34), respectively; the expression plasmid was pJF118EH (20).

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The ~-glyceraldehyde-3-phosphate dehydrogenase from the extremely thermophilic archaebacterium Methanothermus fervidus was purified and crystallized. The enzyme is a homomeric tetramer (molecular mass of subunits 45 kDa). Partial sequence analysis shows homology to the enzymes from eubacteria and from the cytoplasm of eukaryotes. Unlike these enzymes, the ~-glyceraldehyde-3-phosphate dehydrogenase from Methanothermus fervidus reacts with both NAD' and NADP' and is not inhlbited by pentalenolactone. The enzyme is intrinsically stable up to 75°C. It is stabilized by the coenzyme NADP' and at high ionic strength up to about 90°C. Breaks in the Arrhenius and Van't Hoff plots indicate conformational changes of the enzyme at around 52°C.Despite numerous studies on enzymes from thermophilic organisms mainly isolated from eubacterial and eukaryotic sources [ 11 the structure principles which enable these enzymes to be stable and active at high temperature remain unclear. Detailed structural comparisons between mesophilic and thermophilic enzyme homologues, especially of D-glyCeraldehyde-3-phosphate dehydrogenase, L-lactate dehydrogenase and ferredoxin [2-51 did not lead to a consensus of structural details responsible for stability and activity at high temperature. In some cases, however, the phylogenetic distances between the mesophilic and thermophilic organisms used in these comparisons may be too large for detecting thermophily-specific structures. Also, the detailed investigations on different temperature-sensitive mutants of tryptophan synthetase, lysozyme and A repressor [6 -81 deal only with the thermostability and thus do not consider the whole spectrum of biochemical adaptations to high temperature.The discovery of extremely thermophilic archaebacteria gives rise to the hope that the enzyme proteins of these organisms could show the structural requisites for the thermophilic behaviour more clearly than the proteins of the rather moderately thermophilic eubacteria and eukaryotes investigated up to now.Thermophilic archaebacteria were found among the methanogens and in the so-called sulfur-dependent group. Within the methanogens closely related mesophilic and thermophilic organisms provide an ideal basis for comparisons between mesophilic and thermophilic enzyme proteins, the sulfur-dependent archaebacteria comprise exclusively thermophilic organisms with Pyrodictium occultum Enzymes. ~-Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12); ~-glyceraldehyde-3-phosphate dehydrogenase (NADP') (phosphorylating) (EC 1.2.1.13); trypsin (EC 3.4.21.4); L-lactate dehydrogenase (EC 1.1.1.27); D-lactate dehydrogenase (EC 1.1.1.28); cytochrome c reductase (EC 1.9.99.1); catalase (EC 1.11.1.6).coccus furiosus [lo] and Pyrococcus woesei [ l l ] as the most thermophilic organisms known as yet.For analyzing the structure and function of thermophilic archaebacterial enzymes we focus on the ~-glyceraldehyde-3-phosphate dehydrogenase because this enzyme, at least the enzyme homologues from mesophilic and thermophilic eubacteria...

The genes (slgA) encoding the surface-layer glycoproteins of the hyperthermophilic methanogens Methanothermus fervidus and Methanothermus sociabilis were cloned and sequenced. The nucleotide sequences of these genes differ at only nine positions, resulting in three amino acid differences.In both organisms, the transcription start site was localized by primer extension analyses. The DNA sequence at this site conforms to the promotor box B motif for promotors of archaea. 24 nucleotides upstream of the transcription start is an A + T-rich region, which closely resembles the consensus box A motif of promoters of methanogens. Ribosome binding sites are exactly complementary to the 3' end of the 16s rRNA of these met hanogens.Both slgA genes encode for a precursor of the mature surface-layer protein containing 593 amino acid residues with a putative N-terminal signal sequence of 22 amino acid residues. The deduced protein sequences contain 20 sequon structures representing possible carbohydrate-binding sites.In comparison with other surface-layer proteins, these obtained from the two hyperthermophilic methanogens contain unusually high amounts of isoleucine, asparagine and cysteine residues. Predicted secondary structures have a high content of fl-sheet structure (44%) and only 7% a-helix structures.Glycosylated proteins fulfil a variety of important functions in eukaryotes [l, 21, whereas in prokaryotes glycoproteins have been found almost exclusively in the cytoplasmic membrane or in the surface layers [3 -61.The surface layers consist of protein or glycoprotein subunits, which assemble into mono-layered crystalline arrays. Surface layers are widespread among bacteria and archaea and they can be part of complex cell walls or they can form the only cell wall layer. Surface layers are involved in maintaining shape, cell protection and cell adhesion and they function as molecular barriers and sieves.In extremely thermophilic bacteria, surface layers are directly exposed to the extreme environments and cannot be stabilized by chemical components as is the case with cytoplasmatic proteins [7]. The molecular basis for stability, heat resistance and evolution of the extremely thermophilic surface layer (glyco-) The genes encoding surface-layer proteins have been sequenced from four prokaryotes [8 -1 ll. Recently, we reported on the biosynthesis of the glycan strands of the surface-layer glycoprotein of the methanogen Methanothermus fervidus [ 12, 131. In this paper we describe the cloning and sequencing of the encoding genes of the surface layer glycoproteins of the hyperthermophilic methanogens M t . fervidus and Methanothermus sociabilis in order to obtain more information on the primary structure of extremely thermophilic surface-layer glycopro teins. [19]. Restriction enzymes were purchased from Boehringer Mannheim o r from BRL. The M-MuLV reverse transcriptase, DNase I and the universal and reverse primer for sequencing were from Boehringer Mannheim; T4 DNA ligase and exonuclease BAL31 from BRL; and exonuclease 111 a...