Molecular characterization of the flowering time gene FRIGIDA in Brassica genomes A and C

An important determinant of flowering time variation in Arabidopsis, the FRIGIDA (FRI) gene has not been until recently investigated in economically important Brassica species. In diploid Brassica species, this gene exists as two paralogous loci on chromosomes A3 and A4 (B. rapa; A genome), and C3 and C9 (B. oleracea; C genome). Each locus is represented by several genome-specific alleles, which are discerned primarily by polymorphisms in C- and especially N-terminal regions. Locus- and genome-specific sequences of two FRI paralogues are conserved almost completely in the subgenomes A and C of tetraploid B. napus. The phylogenetic analysis of available FRI sequences presumes that the duplication of FRI loci preceded speciation in the genus Brassica.     

大黄鱼Flotillin-1基因分子特征分析

浮舰蛋白-1(Flotillin-1)是属于SPFH家族的蛋白,是重要的脂筏标志性蛋白.在构建大黄鱼(Larimichthys crocea)肌肉组织cDNA文库的基础上,克隆了Flotillin-1基因,并进一步扩增出内含子.克隆到的序列全长为2497 bp,其中编码区1194 bp,编码397个氨基酸.生物信息学分析大黄鱼Flotillin-1有5类20个功能位点,存在2次跨膜结构,N端和C端都位于细胞膜内.大黄鱼Flotillin-1氨基酸序列具有非常高的保守性,与大西洋鲑和斑马鱼的同源性都在80%以上.在组织中的表达也非常广泛,其中在脾中的表达最强.     

Molecular characterization of the human PLC β1 gene

Inositide-specific phospholipase C (PLC) signaling constitutes a central intermediate in a number of cellular functions among which the control of cell growth raises a particular interest. Indeed, we have previously shown that nuclear phospholipase C β1 (PLC β1) is central for the regulation of mitogen-induced cell growth. We have also assigned by fluorescence in situ hybridization (FISH) analysis the PLC β1 to human chromosome 20p12. In this study, we have carried out a detailed analysis of the human gene, showing the existence of alternative splicing, which gives rise, besides the two forms (1a and 1b) already shown in rodents, to a new 600 bp smaller form coding for a 110 kDa protein. We have also identified a new exon at the 5′, showing no homology with the rodent sequence. Here we provide the complete determination of the exon/intron structure of the gene spanning 250 kb of DNA. We found that the exons are quite small, ranging from 49 to 222 bp, while the introns vary between 108 bp and 34,400 bp. The availability of the understanding of the genome organization of this inositide-specific PLC, which represents a key step of the cell cycle related signaling, could actually pave the way for further genetic analysis of p12 region of human chromosome 20 in diseases involving alterations of the control of cell growth such as malignancies.     

Molecular characterization of the human PLC beta1 gene

Inositide-specific phospholipase C (PLC) signaling constitutes a central intermediate in a number of cellular functions among which the control of cell growth raises a particular interest. Indeed, we have previously shown that nuclear phospholipase C beta1 (PLC beta1) is central for the regulation of mitogen-induced cell growth. We have also assigned by fluorescence in situ hybridization (FISH) analysis the PLC beta1 to human chromosome 20p12. In this study, we have carried out a detailed analysis of the human gene, showing the existence of alternative splicing, which gives rise, besides the two forms (1a and 1b) already shown in rodents, to a new 600 bp smaller form coding for a 110 kDa protein. We have also identified a new exon at the 5', showing no homology with the rodent sequence. Here we provide the complete determination of the exon/intron structure of the gene spanning 250 kb of DNA. We found that the exons are quite small, ranging from 49 to 222 bp, while the introns vary between 108 bp and 34,400 bp. The availability of the understanding of the genome organization of this inositide-specific PLC, which represents a key step of the cell cycle related signaling, could actually pave the way for further genetic analysis of p12 region of human chromosome 20 in diseases involving alterations of the control of cell growth such as malignancies.     

Gibberellins in relation to flowering in Polianthes tuberosa

Endogenous gibberellins (GAs) in corms of Polianthes tuberosa L. (cv. Double) were isolated and identified by high performance liquid chromatography, bioassay and combined capillary gas chromatography-mass spectrometry (GC-MS). Gibberellins A₁, A₁₉, A₂₀ and A₅₃ were quantified at the vegetative, early floral initiation and flower development stages. The identification of 13-hydroxylated GAs indicates the presence of the early 13-hydroxylation pathway in P. tuberosa corms. An increase in GA₁ and GA₂₀, and a decrease in GA₁₉ levels, coincided with the transition from the vegetative phase to the stages of early floral initiation and flower development. GA₅₃ stayed at constant levels at the 3 different growth stages. The absence of GA₁ in vegetative corms and its presence in corms at early floral initiation and flower development stages suggest that GA₁ is a causal factor in inducing floral initiation in P. tuberosa . When GA₁, GA₃, GA₄, GA₂₀ and GA₃₂ were applied to corms at the vegetative stage (plants about 5 cm in height), floral initiation was promoted by all of the GAs used, GA₃₂ being the most active. In contrast with the other GAs, GA₃₂ had no effect on stem elongation. Therefore, it is suggested that hydroxylated C-19 GAs play an important role in flower induction in P. tuberosa .     

Molecular characterization of the PpMADS1 gene from peach

PpMADS1, a member of the euAP1 clade of the class A genes, was previously cloned from peach. In this study, PpMADS1 was constitutively expressed in Arabidopsis thaliana to study its function in plant development. The transgenic A. thaliana plants containing 35S::PpMADS1 showed severe phenotype variation including early flowering, conversion of inflorescence branches to solitary flowers, formation of terminal flowers, production of higher number of carpels, petals, and stamens than non-transgenic plants, and prevention of pod shatter. Significantly, the transgenic plants produced more than one silique from a single flower. The results obtained by using cDNA microarray and real-time PCR analyses in the transgenic Arabidopsis indicated that PpMADS1 might play dual roles in regulating the floral meristem development by activating or repressing different sets of genes that would determine the different fate of a floral meristem. In addition, the PpMADS1 gene promoter was further cloned, and deletion analyses were conducted by using fused GUS as a reporter gene in transgenic A. thaliana. Histochemical staining of different organs from transgenic plants revealed the region between ?197 and ?454?bp was specific for GUS expression in flower primordium, and the region between ?454 and ?678?bp was specific for GUS expression in sepals and petals. In contrast, a negative regulatory element present between ?678 and ?978?bp could suppress GUS expression in filament.     

Molecular cloning and characterization of a gene regulating flowering time from Alfalfa (Medicago sativa L.)

Genes that regulate flowering time play crucial roles in plant development and biomass formation. Based on the cDNA sequence of Medicago truncatula (accession no. AY690425), the LFY gene of alfalfa was cloned. Sequence similarity analysis revealed high homology with FLO/LFY family genes of other plants. When fused to the green fluorescent protein, MsLFY protein was localized in the nucleus of onion (Allium cepa L.) epidermal cells. The RT-qPCR analysis of MsLFY expression patterns showed that the expression of MsLFY gene was at a low level in roots, stems, leaves and pods, and the expression level in floral buds was the highest. The expression of MsLFY was induced by GA₃ and long photoperiod. Plant expression vector was constructed and transformed into Arabidopsis by the agrobacterium-mediated methods. PCR amplification with the transgenic Arabidopsis genome DNA indicated that MsLFY gene had integrated in Arabidopsis genome. Overexpression of MsLFY specifically caused early flowering under long day conditions compared with non-transgenic plants. These results indicated MsLFY played roles in promoting flowering time.     

Molecular cloning and characterization of the obg gene of Streptomyces griseus in relation to the onset of morphological differentiation.

Morphological differentiation in microorganisms is usually accompanied by a decrease in intracellular GTP pool size, as has been demonstrated in bacillaceae, streptomycetaceae, and yeasts. The obg gene, which codes for a GTP-binding protein belonging to the GTPase superfamily of proteins, was cloned from Streptomyces griseus IFO13189. The gene is located just downstream of the genes for ribosomal proteins L21 and L27, encoded a protein of 478 amino acids (51 kDa), and possessed three consensus motifs which confer GTP-binding ability; Obg protein expressed in Escherichia coli bound GTP, as demonstrated using a UV cross-linking method. Introduction of multiple copies of obg into wild-type S. griseus suppressed aerial mycelium development in cells on solid media. However, no effect on streptomycin production was detected, indicating that Obg is involved in the regulation of the onset of morphological but not physiological differentiation. Multiple copies of obg also suppressed submerged spore formation in liquid culture. Southern hybridization studies indicated that genes homologous to obg exist widely in streptomycetes, and an obg homolog was successfully cloned from S. coelicolor A3(2). We propose that by monitoring the intracellular GTP pool size, the Obg protein is involved in sensing changes in the nutritional environment leading ultimately to morphological differentiation.     

Molecular cloning and characterization of a new gene, Oocyte-G1

Oocytes are recognized as a source of regulatory molecules that influence follicular development through an array of actions on granulosa cells. Recently, more and more hormones and signaling molecules were identified during follicular developmental processes; however, the details about their functions are still unclear. During efforts to clone follicular development-related genes, we isolated a cDNA fragment by DDRT-PCR. To obtain cDNA 5'- and 3'-end sequences, we screened a mouse ovarian cDNA library. After screening the library, an open reading frame of 2,994 bp for the new gene (Oocyte-G1), which encodes a 997-residue protein, was cloned. Northern blot analysis revealed the presence of approximately 3.6 kb Oocyte-G1 mRNA in ovary, lung, kidney, testis and brain. Northern analysis of RNA from ovaries in vivo showed that Oocyte-G1 was weakly expressed on day 5 and at a moderate level on day 10. Thereafter, on day 15 or in adults (day 40), there was an increase in expression, followed by a decline in ovaries on day 20 or older (day 120). Furthermore, we studied the Ooctye-G1 protein by using the antiserum against a peptide sequence unique to this gene in Western blotting and immunolocalization. The antiserum recognized a prominent band of approximately 110 kDa in immunoblots and signals were dispersed in oocytes and some cumulus granulosa cells. Our results suggest the potential role of Oocyte-G1 in ovarian follicular development.     

Molecular genetics of the apolipoprotein B gene in pigs in relation to atherosclerosis

Immunologically defined alleles of the pig apolipoprotein B (ApoB) locus (apoB) are correlated with different blood cholesterol levels and predisposition towards premature coronary heart disease. We show here that these alleles are associated with differences in the apoB gene by identifying six restriction fragment length polymorphisms at the pig apoB locus. We have sequenced a 2.4-kb fragment encompassing exons 11 through 14 of one allele, and 7.1 kb from the 3' one-third of exon 26 to about 1 kb past the last exon from another allele. The decoded amino acids of the pig and human ApoB proteins are identical at 70% of these positions. One region close to the C-terminus of the protein is surprisingly different in pigs and humans (57% identity) but the C-terminal region is relatively well conserved (74% identity). Neither of the two putative low-density lipoprotein (LDL) receptor-binding sites is completely conserved in pigs and humans, but identical stretches of amino acids occur near these sites more frequently than in the other sequenced regions. We compare the nucleotide sequences of the region encompassing the putative LDL receptor-binding sites from four pig alleles, including one implicated directly in atherosclerosis. None of the differences appears to account for the hypercholesterolemic phenotype. We conclude that significant differences in the physiology of LDL particles result from changes outside the putative receptor-binding region.     

Cloning and characterization of a flowering time gene from Thellungiella halophila

Thellungiella halophila (T. halophila) (salt cress) is a close relative of Arabidopsis and a model plant for salt tolerance research. However, the nature of its later flowering causes some difficulties in genetic analysis. The FRIGIDA (FRT) gene plays a key role in the Arabidopsis vernalization flowering pathway, whose homolog in T. halophila may also be a key factor in controlling flowering time. In order to study the molecular mechanism of vernalization responses in T. halophila , a full length cDNA named ThFRI (Thellungiella halophila FRIGIDA) was isolated from the young seedlings of T. halophila by RT-PCR and RACE. The ThFRI cDNA was 2017 bp in length and contained an open reading frame encoding a putative protein of 605 ami no acids. The ThFRI showed significant homology to AtFRI (74.5% at the nucleotide level and 63.9% at the ami no acid level). To study its function, ThFRI cDNA was transformed into Arabidopsis thaliana , driven by CaMV 35S promoter. Transgenic plants expressing ThFRI exhibited late-flowering phenotype, which suggests that ThFRI is the funtional FRI homolog in T. halophila . The cloning and funtional characterization of the FRI homolog of T. halophila will faciliate further study of flowering time control in T. halophila .     

Molecular characterization of Bu-1 and TLR2 gene in Haringhata Black chicken

Haringhata Black is the only registered indigenous poultry genetic resource of West Bengal till date. Molecular characterization of HB revealed that Bu-1 to be highly glycoylated transmembrane protein unlike mammalian Bu-1, whereas TLR2 of HB chicken was observed to be rich in Leucine rich repeat. HB chicken was observed to be genetically close to chicken of Japan, while distant to chicken breed of UK and Chicago. Avian species wise evolution study indicates genetic closeness of HB chicken with turkey. Differential mRNA expression profile for the immune response genes (TLR2, TLR4 and Bu1 gene) were studied for HB chicken with respect to other chicken breed and poultry birds, which reveals that HB chicken were better in terms of B cell mediated immunity and hence better response to vaccination. Hence HB chicken is one of the best poultry genetic resources to be reared under backyard system where biosecurity measures are almost lacking.