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1.
Ligation-mediated suppression-PCR as a powerful tool to analyse nuclear gene sequences in the green alga Chlamydomonas reinhardtii 总被引:1,自引:0,他引:1
To improve the analysis of unknown flanking DNA sequences adjacent to known sequences in nuclear genomes of photoautotrophic
eukaryotic organisms, we established the technique of ligation-mediated suppression-PCR (LMS-PCR) in the green alga Chlamydomonas reinhardtii for (1) walking from a specific nuclear insertion fragment of random knockout mutants into the unknown flanking DNA sequence to identify and analyse disrupted genomic DNA regions and for (2) walking
from highly conserved DNA regions derived from known gene iso-forms into flanking DNA sequences to identify new members of
protein families. The feasibility of LMS-PCR for these applications was successfully demonstrated in two different approaches.
The first resulted in the identification of a genomic DNA fragment flanking a nuclear insertion vector in a random knockout mutant whose phenotype was characterised by its inability to perform functional LHC state transitions. The second approach
targeted the cab gene family. An oligonucleotide of a cabII gene, derived from a highly conserved region, was used to identify
potential cab gene regions in the nuclear genome of Chlamydomonas. LMS-PCR combined with 3′ rapid amplification of cDNA ends (3′ RACE) and a PCR-based screening of a cDNA library resulted in the identification of the new cabII gene lhcb4. Both results
clearly indicate that LMS-PCR is a powerful tool for the identification of flanking DNA sequences in the nuclear genome of
Chlamydomonas reinhardtii.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
2.
R. P. Funke J. L. Kovar J. M. Logsdon Jr. J. C. Corrette-Bennett D. R. Straus D. P. Weeks 《Molecular & general genetics : MGG》1999,262(1):12-21
Acetolactate synthase (ALS) catalyzes the first committed step in the synthesis of branched-chain amino acids. In green plants
and fungi, ALS is encoded by a nuclear gene whose product is targeted to plastids (in plants) or to mitochondria (in fungi).
In red algae, the gene is plastid-encoded. We have determined the complete sequence of nucleus-encoded ALS genes from the
green algae Chlamydomonas reinhardtii and Volvox carteri. Phylogenetic analyses of the ALS gene family indicate that the ALS genes of green algae and plants are closely related,
sharing a recent common ancestor. Furthermore, although these genes are clearly of eubacterial origin, a relationship to the
ALS genes of red algae and cyanobacteria (endosymbiotic precursors of plastids) is only weakly indicated. The algal ALS genes
are distinguished from their homologs in higher plants by the fact that they are interrupted by numerous spliceosomal introns;
plant ALS genes completely lack introns. The restricted phylogenetic distribution of these introns suggests that they were
inserted recently, after the divergence of these green algae from plants. Two introns in the Volvox ALS gene, not found in the Chlamydomonas gene, are positioned precisely at sites which resemble “proto-splice” sequences in the Chlamydomonas gene.
Received: 27 November 1998 / Accepted: 21 April 1999 相似文献
3.
Beta-tubulin, one of the cytoskeletal proteins, has been highly conserved throughout the evolution of eukaryotes. Degenerate
PCR and inverse PCR (iPCR) were used to isolate the full-length beta-tubulin gene and its 5′ and 3′-flanking regions (2799bp)
from the marine red alga Porphyra yezoensis. This gene, designated as TubB1, is devoid of introns. The canonical cis-acting elements such as TATA box, CAAT box and polyA signal AAUAAA are not found
in flanking sequences, but another putative polyA signal CAYTG is found downstream of the stop codon. Comparison of the deduced
458 amino acid sequences shows higher similarity to the Protoflorideophycidae Cyanidioschyzon merolae (82%) than to the red alga Chondrus crispus (79%). Codon bias indicates strong expression of TubB1. Phylogenetic analysis suggests that the beta-tubulin of P. yezoensis and C. merola go together with fungi and not with green plants. These nucleotide sequence data have been deposited in the DDB/EMBL/Genbank
databases under the accession number AY221630. 相似文献
4.
5.
Xiaoping Chen Zhangying Wang Jianhua Wang Maoyan Wang Li Zhao Guoying Wang 《Plant Cell, Tissue and Organ Culture》2007,88(1):11-20
ADP-glucose pyrophosphorylase (AGPase) represents a key regulatory step in starch synthesis. A 0.9 kb of 5′ flanking region
preceding Brittle2 gene, encoding the small subunit of maize endosperm AGPase, was cloned from maize genome and its expression pattern was studied
via the expression of β-glucuronidase (GUS) gene in transgenic tobacco. Analysis of GUS activities showed that the 0.9 kb
fragment flanking Brittle2 gene was sufficient for driving the seed-preferred expression of the reporter gene. The activity of the 0.9 kb 5′ flanking
fragment was compared with that of the tandem promoter region from a zein gene (zE19, encoding a maize 19 kDa zein protein). The results indicated that both promoters were seed-preferred in a dicotyledonous
system as tobacco and the activity of zE19 promoter was three to fourfold higher than that of the 0.9 kb fragment flanking Brittle2 gene in transgenic tobacco seeds. At the same time, zE19-driven GUS gene expressed earlier than Brittle2 promoter during seed development. Histochemical location of GUS activity indicated that both promoters showed high expression
in embryos, which is different from similar promoters tested in maize. 相似文献
6.
7.
Conservation in wheat high-molecular-weight glutenin gene promoter sequences: comparisons among loci and among alleles of the GLU-B1-1 locus 总被引:2,自引:0,他引:2
O. D. Anderson F. A. Abraham-Pierce A. Tam 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,96(5):568-576
The high-molecular-weight glutenin (HMW) genes and encoded subunits are known to be critical for wheat quality characteristics
and are among the best-studied cereal research subjects. Two lines of experiments were undertaken to further understand the
structure and high expression levels of the HMW-glutenin gene promoters. Cross hybridizations of clones of the paralogous
x-type and y-type HMW-glutenin genes to a complete set of six genes from a single cultivar showed that each type hybridizes
best within that type. The extent of hybridization was relatively restricted to the coding and immediate flanking DNA sequences.
Additional DNA sequences were determined for four published members of the HMW-glutenin gene family (encoding subunits Ax2*, Bx7, Dx5, and Dy10) and showed that the flanking DNA of the examined genes diverge at approximately −1200 bp 5′ to the start
codon and 200–400 bp 3′ to the stop codon. These divergence sites may indicate the boundaries of sequences important in gene
expression. In addition, promoter sequences were determined for alleles of the Bx gene (Glu-B1-1), a gene reported to show higher levels of expression than other HMW-glutenin genes and with variation among cultivars. The
sequences of Bx promoters from three cultivars and one wild tetraploid wheat indicated that all Bx alleles had few differences
and contained a duplicated portion of the promoter sequence “cereal-box” previously suspected as a factor in higher levels
of expression. Thus, the “cereal-box” duplication preceeded the origin of hexaploid wheat, and provides no evidence to explain
the variations in Bx subunit synthesis levels. One active Bx allele contained a 185-bp insertion that evidently resulted from
a transposition event.
Received: 5 August 1997 / Accepted: 6 November 1997 相似文献
8.
The 5′ untranslated regions (UTR) of chloroplast mRNAs often contain regulatory sequences that control RNA stability and/or
translation. The petD chloroplast mRNA in Chlamydomonas reinhardtii has three such essential regulatory elements in its 362-nt long 5′ UTR. To further analyze these elements, we compared 5′
UTR sequences from four Chlamydomonas species (C. reinhardtii, C. incerta, C. moewusii and C. eugametos) and five independent strains of C. reinhardtii. Overall, these petD 5′ UTRs have relatively low sequence conservation across these species. In contrast, sequences of the three regulatory elements
and their relative positions appear partially conserved. Functionality of the 5′ UTRs was tested in C. reinhardtii chloroplasts using β-glucuronidase reporter genes, and the nearly identical C. incerta petD functioned for mRNA stability and translation in C. reinhardtii chloroplasts while the more divergent C. eugametos petD did not. This identified what may be key features in these elements. We conclude that these petD regulatory elements, and possibly the corresponding trans-acting factors, function via mechanisms highly specific and surprisingly sensitive to minor sequence changes. This provides
a new and broader perspective of these important regulatory sequences that affect photosynthesis in these algae. 相似文献
9.
Background
Green algae of the family Volvocaceae are a model lineage for studying the molecular evolution of multicellularity and cellular differentiation. The volvocine alga Gonium is intermediate in organizational complexity between its unicellular relative, Chlamydomonas, and its multicellular relatives with differentiated cell types, such as Volvox. Gonium pectorale consists of ~16 biflagellate cells arranged in a flat plate. The detailed molecular analysis of any species necessitates its accessibility to genetic manipulation, but, in volvocine algae, transformation procedures have so far only been established for Chlamydomonas reinhardtii and Volvox carteri. 相似文献10.
Qian-Jin Zhou Hong-Li Zhang Xiao-Lei Jiang Ai-Fang Du 《Functional & integrative genomics》2010,10(4):589-601
11.
Myostatin (MSTN) is a member of the transforming growth factor-β (TGF-β) superfamily that functions as a negative regulator
of skeletal muscle development and growth in mammals. However, few reports are available about the structure and function
of MSTN in teleost. Here, the MSTN gene was cloned from sea perch (Lateolabrax japonicus) by homology cloning and genomic walking. In the 4873-bp genomic sequence, three exons, two introns, and 5′ and 3′ flanking
sequences were identified. The sea perch MSTN gene encodes a 374-amino acid protein, including a signal peptide, conserved cysteine residues, and a RXXR proteolytic cleavage
domain. Expression analysis of MSTN revealed that MSTN was highly expressed in eyes, brain, and muscle; intermediately in intestine; and weakly in gill, spleen, liver, and heart.
It was demonstrated that MSTN mRNA was highly expressed in embryonic stem cell line (LJES1), but it was undetectable in several
types of somatic cell lines from sea perch, including fibroblast-like cell, epithelioid cell, and lymphocyte-like cell. Further,
it was demonstrated that the 5′ flanking region of the MSTN gene can drive the expression of green fluorescent protein (GFP) reporter gene in LJES1 cells and transgenic zebrafish (Danio rerio). This is the first report on the expression profile of MSTN gene in various types of cell cultures. 相似文献
12.
13.
Vladimir T. Valkov Daniela Gargano Carmela Manna Gelsomina Formisano Philip J. Dix John C. Gray Nunzia Scotti Teodoro Cardi 《Transgenic research》2011,20(1):137-151
Transformation of potato plastids is limited by low transformation frequencies and low transgene expression in tubers. In
order to improve the transformation efficiency, we modified the regeneration procedure and prepared novel vectors containing
potato flanking sequences for transgene integration by homologous recombination in the Large Single Copy region of the plastome.
Vector delivery was performed by the biolistic approach. By using the improved regeneration procedure and the potato flanking
sequences, we regenerated about one shoot every bombardment. This efficiency corresponds to 15–18-fold improvement compared
to previous results with potato and is comparable to that usually achieved with tobacco. Further, we tested five promoters
and terminators, and four 5′-UTRs, to increase the expression of the gfp transgene in tubers. In leaves, accumulation of GFP to about 4% of total soluble protein (TSP) was obtained with the strong
promoter of the rrn operon, a synthetic rbcL-derived 5′-UTR and the bacterial rrnB terminator. GFP protein was detected in tubers of plants transformed with only four constructs out of eleven. Best results
(up to approximately 0.02% TSP) were achieved with the rrn promoter and rbcL 5′-UTR construct, described above, and another containing the same terminator, but with the promoter and 5′-UTR from the
plastid clpP gene. The results obtained suggest the potential use of clpP as source of novel regulatory sequences in constructs aiming to express transgenes in amyloplasts and other non-green plastids.
Furthermore, they represent a significant advancement of the plastid transformation technology in potato, of relevance to
its implementation in potato breeding and biotechnology. 相似文献
14.
15.
Flower colour and cytochromes P450 总被引:8,自引:0,他引:8
Yoshikazu Tanaka 《Phytochemistry Reviews》2006,5(2-3):283-291
Flavonoids are major constituents of flower colour. Plants accumulate specific flavonoids and thus every species often exhibits a limited flower colour range. Three cytochromes P450 play critical roles in the flavonoid biosynthetic pathway. Flavonoid 3′-hydroxylase (F3′H, CYP75B) and flavonoid 3′,5′-hydroxylase (F3′5′H, CYP75A) catalyze the hydroxylation of the B-ring of flavonoids and are necessary to biosynthesize cyanidin-(red to magenta) and delphinidin-(violet to blue) based anthocyanins, respectively. Pelargonidin-based anthocyanins (orange to red) are synthesized in their absence. Some species such as roses, carnations and chrysanthemums do not have violet/blue flower colour due to deficiency of F3′5′H. Successful expression of heterologous F3′5′H genes in roses and carnations results in delphinidin production, causing a novel blue/violet flower colour. Down-regulation of F3′H and F3′5′H genes has yielded orange petunia and pink torenia colour that accumulate pelargonidin-based anthocyanins. Flavone synthase II (CYP93B) catalyzes the synthesis of flavones that contribute to the bluing of flower colour, and modulation of FNSII gene expression in petunia and tobacco changes their flower colour. Extensive engineering of the anthocyanin pathway is therefore now possible, and can be expected to enhance the range of flower colours. 相似文献
16.
17.
Molecular phylogenetic analyses of snakeheads (Perciformes: Channidae) using mitochondrial DNA sequences 总被引:1,自引:0,他引:1
Mitochondrial DNA sequences of approximately 1.5 kbp including the NADH dehydrogenase subunit 2 (ND2) gene and its flanking
gene regions were determined for 20 species from the freshwater fish family Channidae and 3 species from Nandidae, Badidae,
and Osphronemidae. Channa orientalis and C. gachua had an approximately 170-bp insertion between the tRNAMet and ND2 genes, where a 5′-half of the insertion was similar to the 5′-end portion of the ND2 gene and a 3′-half was homologous
to the tRNAMet gene. This insertion may thus have originated from a tandem gene duplication that occurred in a common ancestor of these
two sister species. Molecular phylogenetic analyses from different tree-building methods consistently suggested the mutual
monophyly of the African and Asian taxa and the existence of several clades within the Asian taxa, some of which correspond
to distinct morphological features. Our molecular phylogeny clearly supported multiple independent losses of pelvic fins on
Asian lineages in parallel. Divergence time estimation based on some reasonable assumptions without assuming the molecular
clock suggested the early Cretaceous divergence of the African and Asian channids. The results thus support an ancient vicariant
divergence of the African and Asian channids, rather than the more recent dispersal between African and Eurasian continents. 相似文献
18.
Héctor Musto Héctor Romero Helena Rodríguez-Maseda 《Journal of molecular evolution》1998,46(2):159-167
Synonymous codon choices vary considerably among Schistosoma mansoni genes. Principal components analysis detects a single major trend among genes, which highly correlates with GC content in
third codon positions and exons, but does not discriminate among putatively highly and lowly expressed genes. The effective
number of codons used in each gene, and its distribution when plotted against GC3, suggests that codon usage is shaped mainly by mutational biases. The GC content of exons, GC3, 5′, 3′, and flanking (5′+ 3′+ introns) regions are all correlated among them, suggesting that variations in GC content may
exist among different regions of the S. mansoni genome. We propose that this genome structure might be among the most important factors shaping codon usage in this species,
although the action of selection on certain sequences cannot be excluded.
Received: 10 March 1997 / Accepted: 27 June 1997 相似文献
19.
Ronald W. DeBry 《Journal of molecular evolution》1998,46(3):355-360
Sequences were obtained from five species of rodents that are orthologous to an H2a histone pseudogene from Mus musculus. The pseudogene is part of the cluster of replication-dependent histone genes found on Mus musculus chromosome 13. Comparative analysis of these five sequences together with the previously published sequence from M. musculus shows that this gene has likely been a pseudogene throughout the evolution of the genus Mus, while the gene from Rattus norvegicus is likely functional. Three large (>20 bp) deletions were found among the Mus pseudogenes, a feature that is very unusual compared to surveys of processed pseudogenes. In addition, there are two single-base
deletions and one 4-bp insertion among the Mus pseudogenes. The species distributions of one of the large deletions and the 4-bp insertion require either independent insertions
of an identical sequence, independent deletions with identical boundaries, or a deletion followed by precise reintegration
of the original sequence. The evidence favors the hypothesis of multiple deletions with identical boundaries. The ``coding'
regions of the Mus pseudogenes show a much reduced level of among-species variability in the 3′ half of the pseudogene, compared both to the
5′ half and to flanking sequences. This supports a hypothesis that the 3′ end of the pseudogene is the target of frequent
gene conversion by functional H2a genes.
Received: 1 April 1997 / Accepted: 12 June 1997 相似文献
20.
The unicellular green alga Haematococcus pluvialis (Volvocales) is known for the ketocarotenoid astaxanthin (3, 3′-dihydroxy-β, β-carotene-4, 4′-dione) accumulation, which is induced under unfavorable culture conditions. In this work, we used cDNA microarray analysis to screen differentially expressed genes in H. pluvialis under astaxanthin-inductive culture conditions, such as combination of cell exposure to high irradiance and nutrient deprivation. Among the 965 genes in the cDNA array, there are 144 genes exhibiting differential expression (twofold changes) under these conditions. A significant decrease in the expression of photosynthesis-related genes was shown in astaxanthin-accumulating cells (red cells). Defense- or stress-related genes and signal transduction genes were also induced in the red cells. A comparison of microarray and real-time PCR analysis showed good correlation between the differentially expressed genes by the two methods. Our results indicate that the cDNA microarray approach, as employed in this work, can be relied upon and used to monitor gene expression profiles in H. pluvialis. In addition, the genes that were differentially expressed during astaxanthin induction are suitable candidates for further study and can be used as tools for dissecting the molecular mechanism of this unique pigment accumulation process in the green alga H. pluvialis.
Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. 相似文献