Phylogenetic analysis of the Glomeromycota by partial β-tubulin gene sequences

The 3’ end of the β-tubulin gene was amplified from 50 isolates of 45 species in Glomeromycota. The analyses included a representative selection of all families except Pacisporaceae and Geosiphonaceae. Phylogenetic analyses excluded three intron regions at the same relative positions in all species due to sequence and length polymorphisms. The β-tubulin gene phylogeny was similar to the 18S rRNA gene phylogeny at the family and species level, but it was not concordant at the order level. Species in Gigasporaceae and Glomeraceae grouped together but without statistical support. Paralogous sequences in Glomus species likely contributed to phylogenetic ambiguity. Trees generated using different fungal phyla as out-groups yielded a concordant topology. Family relationships within the Glomeromycota did not change regardless if the third codon position was included or excluded from the analysis. Multiple clones from three isolates of Scutellospora heterogama yielded divergent sequences. However, phylogenetic patterns suggested that only a single copy of the β-tubulin gene was present, with variation attributed to intraspecific sequence divergence.     

Phylogenetic relationships within the ‘core’ Laureae (Litsea complex, Lauraceae) inferred from sequences of the chloroplast gene matK and nuclear ribosomal DNA ITS regions

A phylogenetic analysis of the core Laureae (Litsea complex) was conducted using the chloroplast gene matK and nuclear ribosomal DNA ITS sequences to investigate generic relationships and boundaries within the complex. Despite low genetic divergence for matK, rooting of the tree with Sassafras resulted in Iteadaphne as the basal member of the complex and five resolved clades: a Neolitsea clade and then Laurus, Parasassafras, Litsea and Lindera clades in a large polytomy with unresolved Lindera sections plus Umbellularia. A combined analysis of the data (identical to the ITS results) provided a more resolved phylogeny of the Laureae, with four major lineages: the Laurus, Litsea, Lindera and Actinodaphne II clades. These clades also appear to reflect the importance of inflorescence structure and ontogeny within the Laureae, as well as data from cuticular micromorphology, but there was no support for traditional generic characters such as 2- versus 4- celled anthers. As a result, genera such as Actinodaphne, Litsea, Neolitsea and Lindera were polyphyletic in all analyses. Parasassafras was related to Sinosassafras by the matK data, but distant from it in the ITS and combined analyses.     

Phylogenetic and coevolutionary analysis of the β-barrel protein family comprised of mitochondrial porin (VDAC) and Tom40

Beta-barrel proteins are the main transit points across the mitochondrial outer membrane. Mitochondrial porin, the voltage-dependent, anion-selective channel (VDAC), is responsible for the passage of small molecules between the mitochondrion and the cytosol. Through interactions with other mitochondrial and cellular proteins, it is involved in regulating organellar and cellular metabolism and likely contributes to mitochondrial structure. Tom40 is part of the translocase of the outer membrane, and acts as the channel for passage of preproteins during their import into the organelle. These proteins appear to share a common evolutionary origin and structure. In the current study, the evolutionary relationships between and within both proteins were investigated through phylogenetic analysis. The two groups have a common origin and have followed independent, complex evolutionary pathways, leading to the generation of paralogues in animals and plants. Structures of diverse representatives were modeled, revealing common themes rather than sites of high identity in both groups. Within each group, intramolecular coevolution was assessed, revealing a new set of sites potentially involved in structure-function relationships in these molecules. A weak link between Tom40 and proteins related to the mitochondrial distribution and morphology protein, Mdm10, was identified. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

cDNA cloning of β-tubulin gene and organization of tubulin genes in Vigna radiata (mung bean) genome

We isolated an almost full-length cDNA clone containing -tubulin gene from a partial cDNA library of mung bean using chicken cDNA as probe. Cross-hybridization with chicken -tubulin cDNA and positive hybridization-selection and translation of mung bean mRNA established that this clone contains -tubulin sequences. We studied the organization of tubulin genes in mung bean. In this plant tubulin genes are organized in tandem repeats of alternating - and -tubulin genes. The 5.6 kb basis repeat unit which contains both - and -tubulin genes is repeated twenty times per haploid genome.Abbreviations SDS sodium dodecyl sulphate - 1×SSPE 150 mM NaCl, 10 mM NaH₂PO₄ and 1 mM EDTA, pH 7.4     

Molecular cloning and sequence analysis of the ribosomal ‘A’ protein gene from the archaebacterium,Halobacterium halobium

The ribosomal ‘A’ protein gene of Halobacterium halobium has been cloned and the nucleotide sequence of the DNA fragment containing the ‘A’ protein gene has been determined. The amino-acid sequence of the protein deduced from the nucleotide sequence was established from manual sequence analysis of the protein and structural data provided by peptides derived from cleavage of the protein with various proteinases. The ‘A’ protein consisted of 114 amino acids with a molecular weight of 11562 and was characterized mainly by a high amounts of alanine and acidic amino acid in the C-terminal half of the molecule. The coding sequence of the gene was preceded by a predicted Shine-Dalgarno sequence and two terminal codons. There was no intron or insertion sequence in the coding sequence. Following the terminal codon of the ‘A’ gene, there was a structure reminiscent of the Escherichia coli rho-independent terminator. The G + C content of the coding sequence was found to be 71%. Inspection of the codon usage for the ‘A’ gene revealed 85% preference for G or C at the third codon position.     

Cloning of the β2-microglobulin gene in the zebrafish

The ₂-microglobulin (₂m) is a protein found in the serum in a free form and on the cell surface in a form noncovalently associated with the chain of the class I major histocompatibility complex (Mhc) molecules. In mammals, the ₂m-encoding gene (B2m) is found on a chromosome different from the Mhc proper. We have isolated and characterized the B2m gene of the zebrafish, Brachydanio rerio, family Cyprinidae. We obtained both cDNA and genomic clones of the Brre-B2m gene. The cDNA clones contained the entire coding sequence, the entire 3 untranslated (UT) region, and at least part of the 5UT region. The genomic clone contained the entire Brre-B2m gene. The coding sequence specifies 97 amino acid residues of the mature protein so that the zebrafish ₂m is two residues shorter than human and one residue shorter than cattle, fowl, or turkey ₂m (codons at positions 85 and 86 have been deleted in the Brre-B2m. gene). The amino acid and nucleotide sequence similarities between zebrafish and human ₂m (B2m) are 45% and 59%, respectively. Approximately 24% of the positions are invariant and an additional 9% show only conservative substitutions in comparisons which include all known 2m sequences (fish, avian, and mammalian). Most of the conserved positions are in the strands (some 47% of the -strand positions are conserved in the three vertebrate classes). The Brre-B2m gene consists of four exons separated by three introns. All of the introns are considerably shorter than the corresponding introns in the mammalian B2m genes. The coding sequences of the cDNA and the genomic clones are almost identical but the sequences of the 3'UT regions differ at 1.7% of the sites, suggesting that the genes borne by these clones might have diverged at least 0.7 million years (my) ago. In contrast to the human B2m gene, the Brre-B2m gene shows no bias in the distribution of the CpG dinucleotides: the dinucleotides are distributed evenly along the entire available sequence. The haploid genome of the zebrafish contains only one copy of the B2m gene.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L05383 (B2M) and L05384 (B2RG). Correspondence to: J. Klein.     

Monophyletic groups within the Parmeliaceae identified by ITS rDNA, β-tubulin and GAPDH sequences

Phylogenetic relationships within the Parmeliaceae are analysed cladistically on the basis of DNA characters from partial β-tubulin, partial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ITS sequences. 100 taxa representing 73 of the 88 genera currently recognised are included in the analyses. Eight monophyletic groups including two or more genera were identified in the tree calculated from the combined data matrix. Three of the groups cover almost half of the species of the family. The largest and strongest supported group includes seven genera with their distribution centres in the Southern Hemisphere: Almbornia, Chondropsis, Karoowia, Namakwa, Neofuscelia, Xanthomaculina and Xanthoparmelia. The second group is a clade of four essentially tropical genera: Concamerella, Flavoparmelia, Parmotrema and Rimelia. The third large group with strong support is the core of cetrarioid lichens, distributed primarily in cold areas of the Northern Hemisphere. The genus Parmelia sensu Hale is not closely related with most of its segregates. One new combination, Cetrariella commixta, is proposed. Coelopogon abraxas is reported from South America for the first time.     

20-Hydroxyecdysone induces the expression of one β-tubulin gene in Drosophila Kc cells

The expression of 56D and 60C β-tubulin genes has been examined in Drosophila melanogaster Kc cells in response to the insect moulting hormone, 20-hydroxyecdysone (20-OH-E). Northern blots probed with β-tubulin subclones show that the 56D β-tubulin gene encodes a 1.8 kb mRNA whose abundance is not affected by 20-OH-E. The 60C gene probe detects two mRNAs: one of 1.8 kb present in untreated and 20-OH-E-treated cells, and one of 2.6 kb present only in 20-OH-E-treated cells; using a 60C 3′-specific probe, only the 2.6 kb is revealed. Hybrid selection translation experiment demonstrates that a 20-OH-E-inducible mRNA homologous to the 60C gene encodes a β-tubulin subunit (P4); this subunit is the so-called β3-tubulin. Translation of size-fractionated mRNA shows that the 20-OH-E-induced β3-tubulin subunit is encoded, in treated cells, by the 2.6 kb mRNA.     

Carboxyl terminal sequences of β-tubulin involved in the interaction of HMW-MAPs. Studies using site-specific antibodies

After the finding of the involvement of the C-terminal moieties of tubulin subunits in the interaction of MAPs, different studies have focused on the substructure of the binding domains for the different MAPs. Current biochemical evidence point to the role of a low-homology sequence between and -subunits within the conserved region of the C-terminal domain of tubulin, in the binding of AMP-2 and tau. Another line of studies indicates that a site for interaction of the high molecular weight MAPs is located in the variable region defined by the glutamic-rich C-terminus of -tubulin. Here, we report the usefulness of idiotypic site-directed antibodies, produced by immunization with peptides from different -tubulin isoforms, to study both MAP-1 and MAP-2 binding sites on tubulin. On the basis of these results with site-specific antibodies along with previous structural information (Crosset al. 1991, Biochemistry 30: 4362–4366), we propose the role of consensus sequences, from the invariant -tubulin C-terminal domain in the binding of MAP-2 and from the variable domain in the interactions of MAP-1 and MAP-2.Abbreviations HMW-MAPs High Molecular Weight Microtubule Associated Proteins - PC-tubulin Tubulin Purified by the phosphocellulose chromatographic procedure     

Molecular cloning,sequencing and sequence analysis of the fox-2 gene of Neurospora crassa encoding the multifunctional β-oxidation protein

We present the molecular cloning and sequencing of genomic and cDNA clones of the fox-2 gene of Neurospora crassa, encoding the multifunctional β-oxidation protein (MFP). The coding region of the fox-2 gene is interrupted by three introns, one of which appears to be inefficiently spliced out. The encoded protein comprises 894 amino acid residues and exhibits 45% and 47% sequence identity with the MFPs of Candida tropicalis and Saccharomyces cerevisiae, respectively. Sequence analysis identifies three regions of the fungal MFPs that are highly conserved. These regions are separated by two segments that resemble linkers between domains of other MFPs, suggesting a three-domain structure. The first and second conserved regions of each MFP are homologous to each other and to members of the short-chain alcohol dehydrogenase family. We discuss these homologies in view of recent findings that fungal MFPs contain enoyl-CoA hydratase 2 and d-3-hydroxyacyl-CoA dehydrogenase activities, converting trans-2-enoyl-CoA via d-3-hydroxyacyl-CoA to 3-ketoacyl-CoA. In contrast to its counterparts in yeasts, the Neurospora MFP does not have a C-terminal sequence resembling the SKL motif involved in protein targeting to microbodies.     

Evolution of the β-globin gene cluster in man and the primates

The arrangement of primate β-related globin genes has been determined by restriction endonuclease mapping of genomic DNA from species ranging from prosimians to man. The arrangement of the entire ?γγδβ-globin gene cluster in the gorilla and the yellow baboon is indistinguishable from that of man. Restriction site differences between these species are consistent with a surprisingly low overall rate of intergenic DNA sequence divergence of approximately 1% in 5 million years. A new world monkey (owl monkey) has a single γ-globin gene, suggesting that the ^Gγ-^Aγ-globin gene duplication in man is ancient, and occurred about 20 to 40 million years ago. The β-globin gene cluster in the brown lemur, a prosimian, is remarkably short (about 20,000 base-pairs) and contains single ?-, γ- and β-globin genes. The γ- and β-globin genes in this animal are separated by a curious gene containing the 3′ end of a β-globin gene preceded by sequences related to the 5′ end of the ?-globin gene.     

Evolution and expression analysis of the β-glucosidase (GLU) encoding gene subfamily in maize

The maize ??-glucosidase (ZmGLU1) hydrolyzes cytokinin-conjugates for releasing active cytokinins and thus plays important roles in cytokinin regulatory processes. ZmGLU1 belongs to glycosyl hydrolases 1 (GH1) gene family with a large number of members, and the gene function of other homologs remains to be investigated. In this study, 47 Arabidopsis, 34 rice, 31 brachypodium, 28 sorghum and 26 maize GH1 protein sequences were collected and subsequently used to construct a phylogenetic tree by Neighbor-Joining method. ZmGLU1 together with its 7 paralogs and 4 sorghum homologs were assigned into a distinct group (named GLU subfamily) with far evolutionary distance to other GH1 members. None of the Arabidopsis, rice and brachypodium gene falling into this group indicated a recent evolutionary emergence of GLU subfamily in some Poaceae plants after the divergence of Poaceae species. Phylogeny and comparative genome analysis revealed that GLU subfamily members of maize and sorghum evolved from a common ancestor, and expanded independently in each species by several duplications after maize-sorghum split. Ka/Ks analysis showed that purifying selection played important roles in maintenance of similar functions among the maize GLU paralogs. In addition, the similar protein properties and cytokinin-dependent gene expressions further suggested the similar functions of ZmGLUs in cytokinin activation. However, the organ-dependent expression of ZmGLUs exhibited diverse patterns, which might contribute to their diverse roles in cytokinin homeostasis. Taken together, this work put new insights into the evolution and expression of ZmGLU genes, and provided the foundation for future functional investigations.