Functional characterization of β-ketoacyl-CoA synthase genes from Brassica napus L.

Seed-specifically expressed -ketoacyl-CoA synthase genes of Brassica napus (Bn-FAE1.1 genes) were cloned from two cultivars, namely Askari, a high-erucic-acid type, and Drakkar, a low-erucic-acid type. The genes from the two cultivars were found to be nearly identical. They encode proteins of 507 amino acids, the sequences of which differ only at position 282. The Bn-FAE1.1 gene of Askari, unlike that of Drakkar, was functionally expressed in yeast cells suggesting that the single amino acid exchange effects the low erucic acid phenotype at the E1 gene locus. In yeast cells the -ketoacyl-CoA synthase of Askari elongated not only oleoyl but also palmitoleoyl groups as well as saturated acyl groups in such a way that monounsaturated acyl groups of 22 carbons and saturated ones of 26 carbons were formed as main products. A reporter gene fused to the promoter region of the Bn-FAE1.1 gene from Askari showed seed-specific expression in transgenic rapeseed plants. Over-expression of the coding region of the Askari gene in developing seeds of transgenic Drakkar plants resulted in a significant increase in the levels of eicosenoic acid and erucic acid esterified in the seed oil. On the other hand, in transgenic high-erucic-acid rapeseed plants the increase in erucic acid level was at most 60% although the chimeric Bn-FAE1.1 gene was co-expressed with an erucoyl-CoA-specific lysophosphatidate acyltransferase gene enabling trierucoyl glycerol to accumulate in the seed oil.     

Analysis of capsaicinoid biosynthesis pathway genes expression in callus cultures of Capsicum chinense Jacq. cv. ‘Umorok’

The placental tissue of the highly pungent chilli cultivar, Capsicum chinense Jacq. cv. ‘Umorok’, is used as explants for callus induction. Callus cultures were subcultured after every 32 days and growth curves for a period of six consecutive growth cycles were studied till a stable capsaicinoids producing callus cultures were obtained. The capsaicinoids content in placental tissue explants decreased gradually during the first 2 months of culture as the explants dedifferentiated to form friable callus while the biomass and capsaicinoid content did not show much change in the subsequent growth cycles. The maximum callus biomass of 7.8 g freshweight (FW) or 0.56 g dry weight (DW) per culture were obtained on the 24th day of every growth cycle and the maximum average capsaicinoids content (1.6 mg g^?1 FW capsaicin and 0.78 mg g^?1 FW dihydrocapsaicin) were obtained on the 20th day of every growth cycle. To investigate the underlying dynamics for capsaicinoid biosynthesis during callus formation, comparative gene expression analysis of the genes involved in capsaicinoid biosynthesis pathway were also studied by qRT-PCR analysis. When compared with placental tissue, all the studied genes showed reduced expression during callus formation, especially putative aminotransferase (pAMT) and pungent gene 1 (Pun1), which were extensively down regulated from the 3rd month onwards in the callus cultures. Therefore, the present study revealed that the down-regulated expression of mainly two putative genes in capsaicinoid biosynthetic pathway (pAMT and Pun1) resulted in lower accumulation of capsaicinoids in callus cultures compared to placental tissues of fruits.

     

Endosperm-specific demethylation and activation of specific alleles of α-tubulin genes of Zea mays L.

We have investigated the methylation status of the -tubulin genes, and the degree of accumulation of their mRNAs in endosperm, embryo and seedling tissues of Zea mays L. We have found that many of the -tubulin genes are differentially demethylated in the endosperm relative to the embryo and seedling. However, only for tub2 and tub4 could a correlation between DNA demethylation and increased RNA accumulation be detected. By analyzing the inbred lines W64A and A69Y and their reciprocal crosses, we have also identified in the endosperm two -tubulin genes, tub3 and tub4, that are differentially demethylated if transmitted by the maternal germline, but that remain hypermethylated when transmitted by the paternal germline.     

Are the dominant and recessive plant disease resistance genes similar? A case study of rice R genes and Xanthomonas oryzae pv. oryzae races.

The resistance of rice to its bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) has both qualitative and quantitative components that were investigated using three near-isogenic line sets for four resistance (R) genes (Xa4, xa5, xa13, and Xa21) and 12 Xoo races. Our results indicate that these two resistance components of rice plants were associated with the properties of the R genes. The qualitative component of the R genes was reflected by their large effects against corresponding avirulent Xoo races. The quantitative component of the R genes was their residual effects against corresponding virulent races and their epistatic effects, which together could lead to high-level resistance in a race-specific manner. Our results revealed important differences between the different types of R genes. Two R genes, Xa4 and Xa21, showed complete dominance against the avirulent Xoo races and had large residual effects against virulent ones. They acted independently and cumulatively, suggesting they are involved in different pathways of the rice defensive system. The third R gene, xa5, showed partial dominance or additivity to the avirulent Xoo races and had relatively small but significant residual effects against the virulent races. In contrast, xa13 was completely recessive, had no residual effects against the virulent races, and showed more pronounced race specificity. There was a strong interaction leading to increased resistance between xa13 and xa5 and between either of them and Xa4 or Xa21, suggesting their regulatory roles in the rice defensive pathway(s). Our results indicated that high-level and durable resistance to Xoo should be more efficiently achieved by pyramiding different types of R genes.     

Equilibrium frequencies of α-globin genes

A mathematical model has been developed to study the mechanism of the maintenance of variable numbers of α-globin genes in human populations. The model incorporates both selection and unequal crossing-over. The selection is formulated so that a homozygous individual with a double deletion is lethal and a heterozygous individual with a deletion or addition of an α-globin gene in a chromosome has decreased fitness. This differs from the previous models of stabilizing selection studied by Ohta (1981) and Takahata (1981). The effect of random genetic drift on the α-thalassemia polymorphims has also been studied.It has been shown that, although the results obtained are compatible with the observation of the low frequency of triple α-globin loci, it cannot explain the high frequency of single and double deletions in Asian populations. For the latter case, some type of heterozygote advantage may be operating.     

MUC genes: a superfamily of genes? Towards a functional classification of human apomucins

The MUC genes encode epithelial mucins. Eight different human genes have been well characterized, and two others identified more recently. Among them, a family of four genes, expressed in the respiratory and digestive tracts, is clustered to chromosome 11p15.5; and these genes encode gel-forming mucins which are structurally related to the superfamily of cystine-knot growth factors. A second group is composed of three independent genes encoding various isoforms of mucins including membrane-bound mucins associated to carcinomas. In this second group, MUC3 and MUC4 encode large apomucins containing EGF-like domains.     

Identification of novel α-gliadin genes

Ten novel α-gliadin genes (Gli-ta, Gli-turg1, Gli-turg2, Gli-turg3, Gli-turg4, Gli-turg5, Gli-turg6, Gli-cs1, Gli-cs2, and Gli-cs3) with unique characteristics were isolated from wheat (Triticum aestivumL.), among which Gli-cs1, Gli-cs2, Gli-cs3, and Gli-turg6 were pseudogenes. Gli-cs3 and nine other sequences were much larger and smaller, respectively, than the typical α-gliadins. This variation was caused by insertion or deletion of the unique domain I and a polyglutamine region, possibly the result of illegitimate recombination. Consequently, Gli-cs3 contained 10 cysteine residues, whereas there were 2 cysteine residues only in the other nine sequences. Gli-ta/Gli-ta-like α-gliadin genes are normally expressed during the development of seeds. SDS-PAGE analysis showed that in-vitro-expressed Gli-ta could form intermolecular disulphide bonds and could be chain extenders. A protein band similar in size to Gli-ta has been observed in seed extracts, and mass spectrometry results confirm that the band contains small molecular mass α-gliadins, which is a characteristic of the novel α-gliadins. Mass spectrometry results also indicated that the two cysteine residues of Gli-ta/Gli-ta-like proteins participated in the formation of intermolecular disulphide bonds in vivo.     

Developmental biochemistry of cottonseed embryogenesis and germination. XIX. Sequences and genomic organization of the α globulin (vicilin) genes of cottonseed

The globulin storage protein genes of cotton are found to exist as gene tandems that contain a gene from each of the 2 globulin subfamilies separated by a spacer region of about 2700 or 3400 base pairs. Three different tandems have been identified by restriction endonuclease mapping of genomic DNA. A cDNA that is different from the genes of the tandems in map sites and/or in nucleotide sequence indicates that a fourth tandem probably exists in the cotton genome. Since the species of cotton used here (Gossypium hirsutum) is an amphidiploid, it is likely that two of the tandems are contributed from each genome.Considerable divergence in nucleotide sequence (18%) and in derived amino acid sequence (28%) is found when the 2 genes of a sequenced tandem are compared. The sequence of the cDNA closely resembles one of the genes in the tandem showing only a 4% divergence in nucleotides and a 4.2% divergence in amino acids. Thus the 2 genes of each tandem represent a relatively ancient gene duplication that has given rise to the two globulin subfamilies of cotton. Only one subfamily has a glycosylation site and the glycosylation of its derived proteins gives rise to the 2 molecular weight sets of globulins seen on gel electrophoresis.Other basic features of these genes and their derived proteins are presented.     

Dicyemid’s dilemma: structure versus genes. The unorthodox structure of dicyemid reproduction

The clear phylogenetic status of the enigmatic Phylum Dicyemida is still uncertain. Their primitive body plan lacks essential metazoan synapomorphies, while genetic data favor a kinship with higher lophotrochozoans. This ultrastructural study increases the confusion about this phylum by presenting an unusual gonad and sperm structure lacking all synapomorphies essential for the various phyla of the lophotrochozoans, either free-living ones or parasites. In Dicyema typus, gonadogenesis is reduced to a single somatic cell, i.e., the infusorigen’s axial cell that functions as a somatic gonadal founder cell as soon as a spermatogonium takes residence in its cytoplasm. The spermiogenic cells resulting therefrom are not connected by intercellular bridges and permanently contain a bundle of microtubules in their cytoplasm, obviously a kind of “dormant” spindle having assembled without centrosomes. Primary spermatocytes develop so-called polycomplexes, multiple synaptinemal complexes. The structure of the sperm is based on a certain kind of somatic cell that has been minimally adapted to function as a sperm. The mature sperm consists in only three organelles: an ovoid nucleus with a somatic chromatin structure, numerous pore complexes and a centrally arranged cluster of coiled tubular structures; a bundle of microtubules embedded into a rim running along the nucleus longitudinal surface, projecting out of the cell like a spear; and a lipid vesicle tightly attached to one pole of the nucleus, touching there the adjacent bundle of microtubules. Immunelectron microscopy confirms the somatic condition of mature sperm, revealing somatic histone H1 immunoreactivity over the nucleus, which can be interpreted as synapomorphy shared with Protozoa, Porifera and Cnidaria. Fertilization occurs as selfing, where the sperm penetrates, “bundle of microtubules first”, a primary oocyte attached vis-à-vis on the other side of the plasma membrane of the infusorigen’s axial cell. This somatic situation points to an ancient evolutionary model rather than to a condition caused by retrogression due to their life as commensals.     

The expression of three desaturase genes of Spirulina platensis in Escherichia coli DH5α – Heterologous expression of Spirulina-desaturase genes

The genes from a cyanobacterium--Spirulina platensis strain C1--that encode the acyl-lipid desaturases (desC, desA and desD) involved in gamma-linolenic (GLA) synthesis have been successfully expressed for the first time in Escherichia coli by employing a pTrcHisA expression system. In this report, the authors describe the expression of the three Spirulina N-terminal 6xHis-desaturases as well as the functional analysis of these recombinant proteins. The gene products of desC, desA and desD have approximate molecular masses of 37, 45, and 47 kDa, respectively. Enzymatic activity measurement of these products was carried out in vivo to demonstrate that (i) the expressed proteins are in functional form, and (ii) the cofactors of the host system can complement the system of Spirulina platensis. The study demonstrated that the gene products of desC and desA catalyzed the reactions in vivo where the enzyme substrates were provided in appropriate concentration. This indicates that the delta9 and delta12 desaturases were expressed in the heterologous host in their active form, and that these two reactions can be carried out in an E. coli host cell using its cofactors system. In contrast, delta6 desaturase activity can be detected only in vitro where electron carriers are provided. This suggests that while this enzyme is expressed in the heterologous host in its active form, its function in vivo is suppressed, as the electron carriers of the host system cannot complement the system of Spirulina platensis.