Sequence of chicken c beta 7 tubulin. Analysis of a complete set of vertebrate beta-tubulin isotypes

In chicken, beta-tubulin is encoded by a family of seven genes. We have now isolated and sequenced overlapping cDNA clones corresponding to gene c beta 7 (previously designated c beta 4'), the only chicken beta-tubulin not previously characterized. The inferred amino acid sequence of c beta 7 tubulin is identical with the class I beta-tubulin isotype found in human, mouse and rat. Moreover, c beta 7 is highly expressed in almost all tissue and cell types in chicken, a pattern similar to those of the genes for class I beta-tubulin isotypes in other vertebrates. Comparison of the complete family of chicken beta-tubulin gene sequences reveals that the heterogeneity of beta-tubulin polypeptides encoded in a higher eukaryote is confined to six distinct beta-tubulin isotypes. Five of these are members of evolutionarily conserved isotypic classes (I to V), whereas the sixth represents a divergent erythroid-specific tubulin whose sequence has not been conserved.     

Intron loss mediated structural dynamics and functional differentiation of the polygalacturonase gene family in land plants

The polygalacturonase (PG) gene family is one of the largest gene families in plants. PGs are involved in various plant development steps. The evolutionary processes accounting for the functional divergence and the specialized functions of PGs in land plants are unclear. Whole sets of PG genes were retrieved from the genome web sites of model organisms in algae and land plants. The number of PG genes was expanded by lineage-specific manner with the biological complexity of the organism. Differentiation of PGs was related with phylogenetic hierarchy such as presence of rhamno-PGs from algae to plants, endo- and exo-PGs in land plants, exo-PGs in flowering plants. Gene structure analysis revealed that land plant PG genes resulted from differential intron gain and loss, with the latter event predominating. Differential intron losses partitioned the PGs into separate clades to be expressed differentially during plant development. Intron position and phase were not conserved between PGs of algae and land plants but conserved among PG genes of land plants from moss to vascular plants, indicating that the current introns in the PGs in land plants appeared after the split between unicellular algae and multicelluar land plants. The results demonstrate that the functional divergence and differentiation of PGs in land plants is attributable to intron losses.     

Genomic organization and evolutionary conservation of plant D-type cyclins

Plants contain more genes encoding core cell cycle regulators than other organisms but it is unclear whether these represent distinct functions. D-type cyclins (CYCD) play key roles in the G1-to-S-phase transition, and Arabidopsis (Arabidopsis thaliana) contains 10 CYCD genes in seven defined subgroups, six of which are conserved in rice (Oryza sativa). Here, we identify 22 CYCD genes in the poplar (Populus trichocarpa) genome and confirm that these six CYCD subgroups are conserved across higher plants, suggesting subgroup-specific functions. Different subgroups show gene number increases, with CYCD3 having three members in Arabidopsis, six in poplar, and a single representative in rice. All three species contain a single CYCD7 gene. Despite low overall sequence homology, we find remarkable conservation of intron/exon boundaries, because in most CYCD genes of plants and mammals, the first exon ends in the conserved cyclin signature. Only CYCD3 genes contain the complete cyclin box in a single exon, and this structure is conserved across angiosperms, again suggesting an early origin for the subgroup. The single CYCD gene of moss has a gene structure closely related to those of higher plants, sharing an identical exon/intron structure with several higher plant subgroups. However, green algae have CYCD genes structurally unrelated to higher plants. Conservation is also observed in the location of potential cyclin-dependent kinase phosphorylation sites within CYCD proteins. Subgroup structure is supported by conserved regulatory elements, particularly in the eudicot species, including conserved E2F regulatory sites within CYCD3 promoters. Global expression correlation analysis further supports distinct expression patterns for CYCD subgroups.     

Intron distribution in Plantae: 500 million years of stasis during land plant evolution

Little is known about the evolution of the intron-exon organization in the more primitive groups of land plants, and the intron distribution among Plantae (glauco-, rhodo-, chloro- and streptophytes) has not been investigated so far. The present study is focused on some key species such as the liverwort Marchantia polymorpha, representing the most ancient lineage of land plants, and the streptophycean green alga Mesostigma viride, branching prior to charophycean green algae and terrestrial plants. The intron distribution of six genes for sugar phosphate metabolism was analyzed including four different glyceraldehyde-3-phosphate dehydrogenases (GAPDH), the sedoheptulose-1,7-bisphosphatase (SBP) and the glucose-6-phosphate isomerase (GPI). We established 15 new sequences including three cDNA and twelve genomic clones with up to 24 introns per gene, which were identified in the GPI of Marchantia. The intron patterns of all six genes are completely conserved among seed plants, lycopods, mosses and even liverworts. This intron stasis without any gain of novel introns seem to last for nearly 500 million years and may be characteristic for land plants in general. Some unique intron positions in Mesostigma document that a uniform distribution is no common trait of all streptophytes, but it may correlate with the transition to terrestrial habitats. However, the respective genes of chlorophycean green algae display largely different patterns, thus indicating at least one phase of massive intron rearrangement in the green lineage. We moreover included rhodophyte and glaucophyte reference sequences in our analyses and, even if the well documented monophyly of Plantae is not reflected by a uniform intron distribution, at least one GPI intron is strictly conserved for 1.5 billion years.     

Occurrence of matK in a trnK group II intron in charophyte green algae and phylogeny of the Characeae

A group II intron containing the matK gene, which encodes a splicing-associated maturase, was found in the trnK (lysine tRNA) exon in the chloroplast genome of the six extant genera of green algae in the family Characeae, which among green algae are the sister group to embryophytes (land plants). The characean trnK intron (～2.5 kilobases [kb]) and matK ORF (～1.5 kb) are comparable in size to the intron and ORF of land plants, in which they are similarly found inserted in the trnK exon. Domain X, a sequence of conserved amino acid residues within matK, occurs in the Characeae. Phylogenetic analysis using maximum likelihood (GTR + I + gamma likelihood model) and parsimony (branch and bound search) yielded one tree with high bootstrap support for all branches. The matK tree was congruent with the rbcL tree for the same taxa. The number and proportion of informative sites was higher in matK (501, 31% of matK sequence) compared to rbcL (122, 10%). Characeae branch lengths were on average more than five times longer for matK compared to rbcL and provided better resolution within the Characeae. These findings along with recent genomic analyses demonstrate that the intron and matK invaded the chloroplast genome of green algae prior to the evolution of land plants.     

Cloning of the PpNHAD1 transporter of Physcomitrella patens, a chloroplast transporter highly conserved in photosynthetic eukaryotic organisms

A cDNA that encodes a transporter from the NHAD family was identified in Physcomitrella patens. Computer-based searches using the amino acid sequence of PpNHAD1 revealed that, in addition to being expressed in flowering plants, highly conserved transporters of this family are expressed in red algae, green algae, mosses, liverworts, and photosynthetic stramenopiles, but not in heterotrophic stramenopiles. A chloroplast transit peptide was detected in PpNHAD1 and in most of the related sequences, indicating that PpNHAD1 is a chloroplast transporter. A PpNHAD1-GFP fusion localized to the chloroplast in Physcomitrella protoplasts, and truncation of the N-terminus of the protein dispersed the fluorescence signal outside the chloroplast. PpNHAD1 did not show functional expression in either yeast or bacterial mutants, but truncated proteins with shorter N-termini, PpNHAD1-1 and PpNHAD1-2, could be functionally expressed in bacteria. PpNHAD1-1 alleviated the Li(+) intolerance of a Na(+)-efflux Escherichia coli mutant at acidic pH values. Both PpNHAD1-1 and PpNHAD1-2 reduced the K(+) requirements of a K(+)-influx E. coli mutant more actively at high pH values. PpNHAD1 seems to be an important transporter that mediates ionic homeostasis in chloroplasts from red algae to flowering plants.     

The chloroplast genome sequence of Chara vulgaris sheds new light into the closest green algal relatives of land plants

The phylum Streptophyta comprises all land plants and six monophyletic groups of charophycean green algae (Mesostigmatales, Chlorokybales, Klebsormidiales, Zygnematales, Coleochaetales, and Charales). Phylogenetic analyses of four genes encoded in three cellular compartments suggest that the Charales are sister to land plants and that charophycean green algae evolved progressively toward an increasing cellular complexity. To validate this phylogenetic hypothesis and to understand how and when the highly conservative pattern displayed by land plant chloroplast DNAs (cpDNAs) originated in the Streptophyta, we have determined the complete chloroplast genome sequence (184,933 bp) of a representative of the Charales, Chara vulgaris, and compared this genome to those of Mesostigma (Mesostigmatales), Chlorokybus (Chlorokybales), Staurastrum and Zygnema (Zygnematales), Chaetosphaeridium (Coleochaetales), and selected land plants. The phylogenies we inferred from 76 cpDNA-encoded proteins and genes using various methods favor the hypothesis that the Charales diverged before the Coleochaetales and Zygnematales. The Zygnematales were identified as sister to land plants in the best tree topology (T1), whereas Chaetosphaeridium (T2) or a clade uniting the Zygnematales and Chaetosphaeridium (T3) occupied this position in alternative topologies. Chara remained at the same basal position in trees including more land plant taxa and inferred from 56 proteins/genes. Phylogenetic inference from gene order data yielded two most parsimonious trees displaying the T1 and T3 topologies. Analyses of additional structural cpDNA features (gene order, gene content, intron content, and indels in coding regions) provided better support for T1 than for the topology of the above-mentioned four-gene tree. Our structural analyses also revealed that many of the features conserved in land plant cpDNAs were inherited from their green algal ancestors. The intron content data predicted that at least 15 of the 21 land plant group II introns were gained early during the evolution of streptophytes and that a single intron was acquired during the transition from charophycean green algae to land plants. Analyses of genome rearrangements based on inversions predicted no alteration in gene order during the transition from charophycean green algae to land plants.     

In Silico Identification and Evolutionary Analysis of Plant <Emphasis Type="Italic">MAPKK6s</Emphasis>

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in plants. Linking upstream MAPK kinase kinase (MAPKKK) to downstream MAPK, MAPK kinase (MAPKK) plays a crucial role in MAPK cascade. MAPKK6 is one member of the MAPKK family. In this study, we have found that plant MAPKK6 genes are widely distributed in different plant species, including moss, seedless vascular plants, gymnosperms, and angiosperms. However, no MAPKK6 can be found in genomes of algae. Analysis of exon–intron organization and intron phase showed that plant MAPKK6s are highly conserved genes during plant evolution. In Physcomitrella patens, Selaginella moellendorffii, and Picea glauca, MAPKK6s exist as multicopy genes. In most high plants, however, MAPKK6s exist as single-copy. Phylogenetic analysis indicated that the occurrence of single-copy of MAPKK6s in high plants is likely because of genomic copy-number loss.     

Cloning and characterization of chloroplast ribosomal protein-encoding genes, rpl16 and rps3, of the marine macro-algae, Gracilaria tenuistipitata

In Gracilaria tenuistipitata, a highly differentiated multicellular member of the marine red algae, Rhodophyta, chloroplast (cp) DNA can be separated as a satellite band from the nuclear DNA in a CsCl gradient. Using a heterologous probe from Chlamydomonas, the ribosomal protein-encoding gene, rpl16, was located on a 4.5-kb EcoRI fragment of cp DNA. The fragment was cloned and a 1365-bp region around rpl16 was sequenced. The gene order around rpl16, 5′ rpl22-rps3-rpl16, is identical to that detected in the chloroplast DNA of liverwort, tobacco and maize. Both the nucleotide sequence and the amino-acid sequence of rpl16 are more conserved than that of rps3. The rpl16 gene contains no intron, a feature which shows more similarity to the unicellular green algae, Chlamydomonas, than the other land plants. Sequences that may form a stable stem-loop structure were detected within the coding sequence of rpl16.     

CHARACTERIZATION OF A cDNA ENCODING GLUTAMINE SYNTHETASE FROM THE MARINE DIATOM SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)

A cDNA-encoding glutamine synthetase (GS) was isolated from the marine diatom Skeletonema costatum (Greville) Cleve by PCR amplification. Nucleic acid and deduced amino acid sequences of the diatom GS were greater than 50% identical to GS from green algae and vascular plants, and phylogenetic analysis established the diatom GS as a member of the GSII gene family. The presence of an N-terminus signal sequence, identified on the basis of sequence similarity with other chloroplast-localized proteins from diatoms, suggests that the encoded GS isoenzyme is localized to the chloroplast. The GS mRNA was present in log-phase cells grown with either nitrate or ammonium as the sole added nitrogen source. Results from Southern blot analysis of genomic DNA suggested that the cDNA isolated in this study was either a member of a small, highly conserved gene family or that there was allelic variation within the region examined. Phylogenetic analyses further indicated that genes encoding GS from the diatom and two species of green algae diverged prior to the gene duplication, to the isoenzymes in vascular plants, supporting the hypothesis that GS isoenzymes in diatoms, green algae, and vascular plants arose through independent evolutionary events.     

Cloning and nucleotide sequence analysis of genes coding for the major chlorophyll-binding protein of the moss Physcomitrella patens and the halotolerant alga Dunaliella salina

Two clones have been isolated from a genomic library of the moss Physcomitrella patens and a cDNA library of the halotolerant green alga Dunaliella salina. The isolates contain genes coding for the major light-harvesting chlorophyll-a/b-binding protein (CAB) in the photosystem II (PSII) light-harvesting complex (LHCII). The 2544-bp insert of the moss genomic clone contains the complete CAB-coding region and 5' and 3' flanking sequences. The coding region contains an intron of 359 bp which is spanned by a pair of 9-bp perfect direct repeats. There are two CCAAT boxes and five enhancer-like elements related to (G)TGGTTTAAA(G) (Weiher et al., 1983) residing in the intron. Comparisons of the moss cab gene with sequences of light-inducible genes of higher plants reveal homologous and repeated sequences similar to the enhancer element in the 5' region upstream from the TATA and CCAAT boxes thought to be responsive to light inducibility. The 1256-bp algal cDNA contains the complete CAB-coding sequence, a 170-bp 5'-nontranslated region, and a 264-bp 3'-nontranslated region. While the overall homology in the nontranslated regions is low between the cab gene of the moss and that of the alga, the 3'-nontranslated regions of the two contain some sequences that are conserved among the cab genes in higher plants. The deduced amino acid sequences of these two clones are highly conserved except for the N-terminal region. Their hydropathic plots are very similar and both possess three hydrophobic segments that are likely alpha-helical transmembrane segments. The proposed CAB transit peptide sequence of the alga is divergent from that of the moss or higher plants, suggesting that they may have evolved from different origins. Southern blot analysis shows that the cab genes in the moss and the alga, as in higher plants, are encoded by a number of homologous genes constituting a multigene family.     

Kinetics of colchicine binding to purified beta-tubulin isotypes from bovine brain.

Tubulin, the constituent protein of microtubules, is an alpha beta heterodimer; both alpha and beta exist in several isotypic forms whose functional significance is not precisely known. The antimitotic alkaloid colchicine binds to mammalian brain tubulin in a biphasic manner under pseudo-first-order conditions in the presence of a large excess of colchicine (Garland, D. L. (1978) Biochemistry 17, 4266-4272). We have studied the kinetics of colchicine binding to purified beta-tubulin isotypes and find that each of the purified beta-tubulin isotypes binds colchicine in a monophasic manner. The apparent on-rate constants for the binding of colchicine to alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers are respectively 132 +/- 5, 30 +/- 2, and 236 +/- 7 M-1 s-1. When the isotypes are mixed, the kinetics become biphasic. Scatchard analysis revealed that the isotypes differ significantly in their affinity constants (Ka) for binding colchicine. The affinity constants are 0.24 x 10(6), 0.12 x 10(6), and 3.31 x 10(6) M-1, respectively, for alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers. Our results are in agreement with the hypothesis that the beta-subunit of tubulin plays a major role in the interaction of colchicine with tubulin. Our binding data raise the possibility that the tubulin isotypes might play important regulatory roles by interacting differently with other non-tubulin proteins in vivo, which in turn, may regulate microtubule-based functions in living cells.     

Comparative analysis of tubulin sequences

1. Information on the structure and evolution of tubulin has been obtained by comparing the available sequence data on 31 alpha-tubulins and 31 beta-tubulins. 2. Similar numbers of conserved amino acids are found amongst both alpha- and beta-tubulins (alpha: 48%, plus conservative substitutions: 72%; beta: 48%, plus conservative substitutions: 70%). About half of them are common to both subunits (23%, plus conservative substitutions: 45%). Four cysteines in the alpha-tubulins and 2 cysteines in the beta-tubulins are conserved. Only one cysteine (position 129) is conserved in all alpha- and beta-tubulins. 3. The longest unbroken stretch of identical amino acids between all the alpha- and beta-tubulins is found in positions 180-186 (Val-Val-Glu-Pro-Tyr-Asn), a region that appears to be important for binding the ribose moiety of GTP. Two other groups of amino acids implicated in GTP binding, one near position 70 and a glycine cluster at position 144 are also quite conserved. 4. Extra length differences between tubulin subunits, presumably present as extensions on the dimer surface, have been observed at position 50 and near position 360 in alpha-tubulins and in one case at position 57 in a beta-tubulin. 5. The introns of tubulin genes, many of them clustered in the first quarter of the tubulin coding region, do not appear to correspond to any particular structural or functional regions. 6. Mutation rates of tubulins vary considerably. The lowest alpha-tubulin homology (62.3%) is between a very divergent Drosophila alpha-tubulin and an alpha-tubulin from the yeast S. cerevisiae. The lowest beta-tubulin homology (63.3%) is between a yeast (S. cerevisiae) beta-tubulin and a mouse beta-tubulin expressed in hematopoietic tissue. In contrast, some mammalian and bird tubulins are almost identical. 7. Tubulin's heterogeneous C-termini are useful for identifying corresponding tubulins of different vertebrate species, many of which are remarkably conserved. Exceptions are the divergent beta-tubulins of erythrocyte and thrombocyte marginal bands. 8. We have proposed a model for tubulin evolution in metazoan organisms in which the release of structural constraints after gene duplication is a major cause of relatively rapid change.     

The sequence and expression of the divergent beta-tubulin in chicken erythrocytes

We report here the complete sequence of a highly divergent chicken erythrocyte beta-tubulin, c beta 6, which appears to represent a major exception to the observation that the primary sequences and sites of expression of beta-tubulin isotypes are conserved within vertebrates. The amino acid sequence was deduced from overlapping cloned cDNAs identified in a chicken erythroblast cDNA library contained in the expression vector, lambda gt11. Compared with other chicken beta-tubulins, among which the maximum sequence divergence is only 8%, c beta 6-tubulin is more hydrophobic, contains seven fewer net negative charges, and exhibits a surprising 17% overall divergence in its amino acid sequence. DNA and RNA blot analyses show that c beta 6-tubulin is present as a single gene copy in the chicken genome and is specifically expressed in the bone marrow. Comparisons of RNA blots and immunoblots of various cells and tissues confirm that this beta-tubulin isotype is contained specifically in erythrocytes and thrombocytes and accounts for 75% of the beta-tubulin mRNA species contained in developing erythroblasts. Interestingly, c beta 6-tubulin exhibits 18% amino acid sequence divergence relative to MB1, the analogous hematopoietic beta-tubulin contained in mouse.     

A ubiquitous beta-tubulin disrupts microtubule assembly and inhibits cell proliferation

Vertebrate tubulin is encoded by a multigene family that produces distinct gene products, or isotypes, of both the alpha- and beta-tubulin subunits. The isotype sequences are conserved across species supporting the hypothesis that different isotypes subserve different functions. To date, however, most studies have demonstrated that tubulin isotypes are freely interchangeable and coassemble into all classes of microtubules. We now report that, in contrast to other isotypes, overexpression of a mouse class V beta-tubulin cDNA in mammalian cells produces a strong, dose-dependent disruption of microtubule organization, increased microtubule fragmentation, and a concomitant reduction in cellular microtubule polymer levels. These changes also disrupt mitotic spindle assembly and block cell proliferation. Consistent with diminished microtubule assembly, there is an increased tolerance for the microtubule stabilizing drug, paclitaxel, which is able to reverse many of the effects of class V beta-tubulin overexpression. Moreover, transfected cells selected in paclitaxel exhibit increased expression of class V beta-tubulin, indicating that this isotype is responsible for the drug resistance. The results show that class V beta-tubulin is functionally distinct from other tubulin isotypes and imparts unique properties on the microtubules into which it incorporates.     

Structural and Biochemical Characterization of Aldehyde Dehydrogenase 12, the Last Enzyme of Proline Catabolism in Plants

Heterokonts, Alveolata protists, green algae from Charophyta and Chlorophyta divisions, and all Embryophyta plants possess an aldehyde dehydrogenase (ALDH) gene named ALDH12. Here, we provide a biochemical characterization of two ALDH12 family members from the lower plant Physcomitrella patens and higher plant Zea mays. We show that ALDH12 encodes an NAD⁺-dependent glutamate γ-semialdehyde dehydrogenase (GSALDH), which irreversibly converts glutamate γ-semialdehyde (GSAL), a mitochondrial intermediate of the proline and arginine catabolism, to glutamate. Sedimentation equilibrium and small-angle X-ray scattering analyses reveal that in solution both plant GSALDHs exist as equilibrium between a domain-swapped dimer and the dimer-of-dimers tetramer. Plant GSALDHs share very low-sequence identity with bacterial, fungal, and animal GSALDHs (classified as ALDH4), which are the closest related ALDH superfamily members. Nevertheless, the crystal structure of ZmALDH12 at 2.2-Å resolution shows that nearly all key residues involved in the recognition of GSAL are identical to those in ALDH4, indicating a close functional relationship with ALDH4. Phylogenetic analysis suggests that the transition from ALDH4 to ALDH12 occurred during the evolution of the endosymbiotic plant ancestor, prior to the evolution of green algae and land plants. Finally, ALDH12 expression in maize and moss is downregulated in response to salt and drought stresses, possibly to maintain proline levels. Taken together, these results provide molecular insight into the biological roles of the plant ALDH12 family.