紫花苜蓿MsMATE1基因克隆及其抵御铁胁迫功能的鉴定

多药和有毒化合物外排转运蛋白MATE能转运金属、激素、次生代谢物等多种底物,因而在植物的生长发育中发挥重要作用。本研究基于紫花苜蓿(Medicago sativa L.)基因组数据和缺铁胁迫的转录组数据,克隆获得紫花苜蓿MsMATE1及pMsMATE1启动子(GenBank登录号分别为MN547958和MT505313)。MsMATE1基因长1470 bp,编码489个氨基酸。系统进化分析表明,紫花苜蓿MsMATE1蛋白属于mate家族,与蒺藜苜蓿MtMATE(XP013453190.1)亲缘关系最近。生物信息学分析表明,MsMATE1具有典型的MATE_like超家族结构域,属于H+势驱动的真核亚类;MsMATE1为跨膜蛋白,二级结构的主要构成元件是α螺旋。pMsMATE1启动子长1598 bp,序列分析显示内含多个植物激素和逆境胁迫响应元件。MsMATE1在紫花苜蓿幼苗的根茎叶中都有表达,茎中的表达量最高。高铁和缺铁胁迫下,MsMATE1基因在紫花苜蓿幼苗各部位的表达显著上调,茎中上调最明显。高铁和缺铁胁迫,转MsMATE1基因烟草的3种抗氧化酶活性、叶绿素和可溶性蛋白含量显著增加,丙二醛含量显著降低。以上结果表明,MsMATE1在烟草中的异源表达提高了植物抵御高铁和缺铁胁迫的能力。MsMATE1可以作为应用基因工程方法改良植物铁胁迫耐受的重要候选基因,本研究为深入了解MsMATE1蛋白在植物响应铁胁迫中的分子机制奠定基础,MsMATE1基因应对其他金属胁迫或环境胁迫的功能有待于进一步鉴定。     

Chromatin structure of Schizosaccharomyces pombe. A nucleosome repeat length that is shorter than the chromatosomal DNA length.

We have used new methods for chromatin isolation, together with conventional methods for measuring the nucleosome repeat length, to determine the repeat length of Schizosaccharomyces pombe chromatin. We obtain a result of 156(+/- 2) bp. Equivalent results are obtained using a psoralen crosslinking method for measuring the repeat length in viable spheroplasts. That result, together with other control experiments, rules out many possible artifacts. The measured value of 156(+/- 2) bp is smaller than the length of DNA found in the chromatosome. Thus, the chromatosome cannot be the fundamental unit of chromatin structure in all eukaryotes. The crossed linker model of chromatin higher order structure is incompatible with a nucleosome repeat length of 156 bp, and thus cannot apply to all eukaryotes. The solenoid model of higher order structure is compatible with this repeat length only if the solenoid is right-handed. We note two other properties of this chromatin. (1) Early in digestion, the DNA length of mononucleosomes from S. pombe and Aspergillus nidulans exceeds the nucleosome repeat length. (2) Many methods for isolating chromatin from S. pombe yield an apparent nucleosome repeat length of less than or equal to 140 bp; this result is found to be an artifactual consequence of nucleosome sliding.     

Cloning and characterization of the gene encoding Aspergillus nidulans DNA topoisomerase II.

We have determined the complete nucleotide sequence of a 5544bp genomic DNA fragment from Aspergillus nidulans that encodes DNA topoisomerase II (topo II). It contains a single open reading frame of 4740bp that codes for 1579 amino acid residues with a molecular weight of 178kDa; when expressed in Escherichia coli and Saccharomyces cerevisiae the molecular weight was 180kDa. The gene (TOP2) is divided into three exons. Two introns, 54bp and 60bp in length, are located at nucleotide positions 187 and 3214 respectively. Comparison of the deduced amino acid sequence with other eukaryotic topo II sequences showed a higher degree of identity with other fungal enzymes than the human topo IIalpha. One of monoclonal antibodies raised against human topo II, 6H8, can cross-react with Aspergillus topo II.     

Tsessebe, Topi and Tiang: three distinct Tc1‐like transposable elements in the malaria vector, Anopheles gambiae

Three distinct types of Tc1‐family transposable elements have been identified in the malaria vector, Anopheles gambiae. These three elements, named Tsessebe, Topi and Tiang, have the potential to encode transposases that retain most of the conserved amino acids that are characteristic of this transposon family. However, all three are diverged from each other by more than 50% at the nucleotide level. Full‐length genomic clones of two types, Topi and Tsessebe, have been isolated and fully sequenced. The third, Tiang, is represented only by a 270 bp, PCR‐amplified fragment of the transposase coding region. The Topi and Tsessebe elements are 1.4 kb and 2.0 kb in length, respectively, and differ in the length of their inverted terminal repeats (ITRs). The Topi elements have 26 bp ITRs, whereas the Tsessebe clones have long ITRs ranging in length from 105 to 209 bp, with the consensus being about 180 bp. This difference is due primarily to variation in the length of an internal stretch of GT repeats. The copy number and location of these elements in ovarian nurse cell polytene chromosomes varies greatly between element subtypes: Topi elements are found at between 17–31 sites, Tsessebe at 9–13 and at 20 euchromatic sites, in addition to several copies of these elements in heterochromatic DNA. The copy number and genomic insertion sites of these transposons varies between A.gambiae strains and between member species of the A.gambiae complex. This may be indicative of transpositionally active Tc1‐like elements within the genome. This revised version was published online in July 2006 with corrections to the Cover Date.     

Human plasminogen activator inhibitor-1 gene. Promoter and structural gene nucleotide sequences

We have determined the nucleotide sequence of the human plasminogen activator inhibitor-1 (PAI-1) gene and significant stretches of DNA which extend into its 5'-and 3'-flanking DNA regions; a total sequence of 15,867 base pairs (bp) is presented. The sequenced 5'-flanking DNA (1,520 bp) contains the essential eukaryotic cis-type proximal regulatory elements CCAAT and TATAA; the more distal 5'-flanking DNA region, as well as some introns, contain sequence elements which share identities with known eukaryotic enhancer elements. A major finding is the identification of a large region of shared nucleotides (comprising of about 520 bp) between the 5'-flanking DNAs of PAI-1 and tissue-type plasminogen activator genes. The length of the PAI-1 5'-untranslated region was found to be 145 bp as determined by nuclease analysis. The remaining PAI-1 structural gene consists of amino acid coding regions (containing a total of 1,206 bp, coding for the 23 amino acids of the signal peptide and 379 amino acids of the mature PAI-1 protein), 8 intron regions (a total of 8,978 bp), and a long 3'-untranslated region of about 1,800 bp which contains several polyadenylation sites. Two types of repetitive DNA elements are located within the PAI-1 structural gene and flanking DNAs: we have found 12 Alu elements and 5 repeats of a long poly (Pur) element. These Alu-Pur elements may represent a subset of the more abundant Alu family of repetitive sequence elements.     

The plasmid replicator AMA1 in Aspergillus nidulans is an inverted duplication of a low-copy-number dispersed genomic repeat

The AMA1 sequence was isolated from a genomic library of Aspergillus nidulans on the basis of its ability to enhance transformation frequency and generate phenotypically unstable transformants in this fungus. These properties were previously shown to be the result of extrachromosomal replication of AMA1-bearing plasmids. Here we demonstrate that AMA1 is an inverted duplication of a sequence which has other isolated genomic copies. These sequences (mobile Aspergillus transformation enhancers, or MATEs) share a high degree of sequence similarity and exhibit some features characteristic of mobile elements, including a potential Met-tRNA priming site, similar to that found in retrotransposons of the Ty- copia group. The nucleotide sequence does not encode any extended polypeptides but contains ARS-consensus matches and a multiply repeated 'Spe' motif, which may be described as a symmetrically duplicated topoisomerase I recognition site. This motif was shown to be a target for illegitimate recombination events. The mobility of members of the MATE family is inferred from the observation that their chromosomal locations are highly variable between wild Aspergillus isolates. The inverted duplication AMA1 is present in laboratory strains derived from the Glasgow isolate but not in other wild isolates tested. This indicates that the inverted duplication AMA1 is of recent evolutionary origin and probably does not exert any conserved function in the chromosome. We discuss possible connections between structural features of AMA1 and its ability to promote extrachromosomal plasmid replication.     

Determination of a functional ancestral sequence and definition of the 5' end of A-type mouse L1 elements

The complete nucleotide sequence of L1Md-A13, a 6372 base-pair (bp) member of the L1Md repetitive family isolated from a BALB/c mouse genomic DNA library, is reported. The nucleotide sequence of 4331 bp from the 5' end of L1Md-9, which is located in the beta-globin complex of the C57BL/10 mouse, is also reported. Parsimony analysis of these sequences plus two previously reported L1Md sequences allows the determination of an ancestral L1Md sequence. Analysis of the L1Md population indicates that this ancestral sequence is likely to represent a functional L1 sequence. This ancestral sequence confirms that the length (1137 bp and 3900 bp) and relationship (14 bp overlap) of the two large open reading frames previously reported are conserved features of the L1Md family. It also allows the determination of an ancestral amino acid sequence for these two open reading frames. Full-length L1Md elements have one of two sequences tandemly repeated at the 5' end. These two monomers are called A-type and F-type. Our data define the 5' end of A-type full-length L1Md elements. L1Md elements of the A-type have varying numbers of tandemly repeated 208 bp monomers, but each element ends about 78 bp from the 5' end of the terminal 208 bp monomer.     

Nucleotide sequence of the phosphoenolpyruvate carboxylase gene of the cyanobacterium Anacystis nidulans

Nucleotide sequence of the open reading frame (ORF) for the phosphoenolpyruvate carboxylase gene (ppc) of the cyanobacterium Anacystis nidulans was determined. The ORF consists of 3159 bp and codes for 1053 amino acid (aa) residues. The codon usage of the ppc of A. nidulans is not so markedly different from that of the Escherichia coli ppc, yet, in A. nidulans the preferred codons are AAG for lysine and CCC for proline, whereas those are seldom used in the E. coli ppc.     

Identification of a major cis-acting DNA element controlling the bidirectionally transcribed penicillin biosynthesis genes acvA (pcbAB) and ipnA (pcbC) of Aspergillus nidulans.

The beta-lactam antibiotic penicillin is produced as a secondary metabolite by some filamentous fungi. In this study, the molecular regulation of the Aspergillus (Emericella) nidulans penicillin biosynthesis genes acvA (pcbAB) and ipnA (pcbC) was analyzed. acvA and ipnA are divergently oriented and separated by an intergenic region of 872 bp. Translational fusions of acvA and ipnA with the two Escherichia coli reporter genes lacZ and uidA enabled us to measure the regulation of both genes simultaneously. A moving-window analysis of the 872-bp intergenic region indicated that the divergently oriented promoters are, at least in part, overlapping and share common regulatory elements. Removal of nucleotides -353 to -432 upstream of the acvA gene led to a 10-fold increase of acvA-uidA expression and simultaneously to a reduction of ipnA-lacZ expression to about 30%. Band shift assays and methyl interference analysis using partially purified protein extracts revealed that a CCAAT-containing DNA element within this region was specifically bound by a protein (complex), which we designated PENR1, for penicillin regulator. Deletion of 4 bp within the identified protein binding site caused the same contrary effects on acvA and ipnA expression as observed for all of the deletion clones which lacked nucleotides -353 to -432. The PENR1 binding site thus represents a major cis-acting DNA element. The intergenic regions of the corresponding genes of the beta-lactam-producing fungi Penicillium chrysogenum and Acremonium chrysogenum also diluted the complex formed between the A. nidulans probe and PENR1 in vitro, suggesting that these beta-lactam biosynthesis genes are regulated by analogous DNA elements and proteins.     

Intron length variation of the Adh gene in Brachyscome (Asteraceae)

Eighteen polymerase chain reaction (PCR) products of the partial sequence of the Adh (alcohol dehydrogenase) gene from 10 Brachyscome species were sequenced and compared. These products contained the 5 three fourths of exon 4 and whole sequences of intron 3. They varied extensively in length due to the differences in length of intron 3. A total of 10 long insertions were flanked by direct repeats of 5 to 12 bp sequences, indicating inserted elements. These inserted elements were classified into the following five categories based on nucleotide sequence characteristics and length; (1) a region homologous to that of 5S RNA genes (5S DNA), (2) A-rich structure at the 3 end-like short interspersed elements (SINEs) in animals, (3) a sequence of 280 bp with no characteristic features, (4) a sequence of 125 bp with no characteristic features, (5) termini of 11 bp inverted repeats flanked by 5 bp sequence of direct repeats characteristics of a transposon.     

The gene for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase is located close to the gene for the large subunit in the cyanobacterium Anacystis nidulans 6301.

The gene for the small subunit (SS) of ribulose-1,5-bisphosphate carboxylase/oxygenase from a cyanobacterium, Anacystis nidulans 6301, has been cloned and subjected to sequence analysis. The SS coding region is located close to and downstream from the large subunit (LS) coding region on the same DNA strand. The spacer region between the LS and the SS coding regions contains 93 base pairs (bp), and has no promoter-like sequences. The coding region of A. nidulans SS gene contains 333 bp (111 codons). The deduced amino acid sequence of the A. nidulans SS protein shows 40% homology with those of higher plants.     

Heterologous transposition in Aspergillus nidulans

Aspergillus nidulans is one of the model ascomycete fungi. Transposition events have never been described in this organism. We have determined that this organism has at least 13 copies of a Fot1-related element. These copies are transcribed, non-methylated and polymorphic in various wild isolates. In spite of this, we have failed to isolate transposon insertions when the resident niaD gene is used as a transposon trap. This contrasts with the situation described previously in Fusarium oxysporum. We show that two elements of F. oxysporum, Fot1 and impala, transpose efficiently in A. nidulans. We have developed the impala system by tagging it with the yA gene. This permits the visual detection of the transposon by the colour of the conidiospores. We demonstrate that no endogenous transposase of A. nidulans is able to act in trans on a defective impala element, whereas its own transposase driven by two different promoters is able to mobilize this element. The frequency of excision of these modified elements is between 10(-4) and 10(-5). Loss of the transposable element occurs in about 10% of all excision events. In the remaining 90%, the transposon seems to be integrated at random positions in the genome. The availability of mitochondrially inherited mutations has allowed us to demonstrate that hybrid dysgenesis is apparently absent in A. nidulans. The development of this system opens the way to investigating the mechanism underlying the paucity of transposition events leading to visible phenotypes. It should allow us to develop efficient gene-tagging tools, useful in this and other fungi.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe: highly homologous introns are inserted at the same position of the otherwise less conserved cox1 genes in Schizosaccharomyces pombe and Aspergillus nidulans.

The DNA sequence of the second intron in the mitochondrial gene for subunit 1 of cytochrome oxidase (cox1), and the 3'' part of the structural gene have been determined in Schizosaccharomyces pombe. Comparing the presumptive amino acid sequence of the 3'' regions of the cox1 genes in fungi reveals similarly large evolutionary distances between Aspergillus nidulans, Saccharomyces cerevisiae and S. pombe. The comparison of exon sequences also reveals a stretch of only low homology and of general size variation among the fungal and mammalian genes, close to the 3'' ends of the cox1 genes. The second intron in the cox1 gene of S. pombe contains an open reading frame, which is contiguous with the upstream exon and displays all characteristics common to class I introns. Three findings suggest a recent horizontal gene transfer of this intron from an Aspergillus type fungus to S. pombe. (i) The intron is inserted at exactly the same position of the cox1 gene, where an intron is also found in A. nidulans. (ii) Both introns contain the highest amino acid homology between the intronic unassigned reading frames of all fungi identified so far (70% identity over a stretch of 253 amino acids). However, in the most homologous region, a GC-rich sequence is inserted in the A. nidulans intron, flanked by two direct repeats of 5 bp. The 37-bp insert plus 5 bp of direct repeat amounts to an extra 42 bp in the A. nidulans intron. (iii) TGA codons are the preferred tryptophan codons compared with TGG in all mitochondrial protein coding sequences of fungi and mammalia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Both positive and negative regulatory elements mediate expression of a photoregulated CAB gene from Nicotiana plumbaginifolia.

We have analyzed promoter regulatory elements from a photoregulated CAB gene (Cab-E) isolated from Nicotiana plumbaginifolia. These studies have been performed by introducing chimeric gene constructs into tobacco cells via Agrobacterium tumefaciens-mediated transformation. Expression studies on the regenerated transgenic plants have allowed us to characterize three positive and one negative cis-acting elements that influence photoregulated expression of the Cab-E gene. Within the upstream sequences we have identified two positive regulatory elements (PRE1 and PRE2) which confer maximum levels of photoregulated expression. These sequences contain multiple repeated elements related to the sequence-ACCGGCCCACTT-. We have also identified within the upstream region a negative regulatory element (NRE) extremely rich in AT sequences, which reduces the level of gene expression in the light. We have defined a light regulatory element (LRE) within the promoter region extending from -396 to -186 bp which confers photoregulated expression when fused to a constitutive nopaline synthase ('nos') promoter. Within this region there is a 132-bp element, extending from -368 to -234 bp, which on deletion from the Cab-E promoter reduces gene expression from high levels to undetectable levels. Finally, we have demonstrated for a full length Cab-E promoter conferring high levels of photoregulated expression, that sequences proximal to the Cab-E TATA box are not replaceable by corresponding sequences from a 'nos' promoter. This contrasts with the apparent equivalence of these Cab-E and 'nos' TATA box-proximal sequences in truncated promoters conferring low levels of photoregulated expression.     

Increased Length of Long Terminal Repeats Inhibits Ty1 Transposition and Leads to the Formation of Tandem Multimers

The Ty1 retrotransposon of Saccharomyces cerevisiae is bounded by long-terminal repeats (LTRs). We have constructed a variety of Ty1 elements in which the LTR length has been increased from the normal length of 334 bp to >2 kb. Although small insertions in the LTR have minimal effects on transposition frequency, larger insertions dramatically reduce it. Nevertheless, elements with long LTRs are incorporated into the genome at a low frequency. Most of these rare insertion events represent Ty1 tandem (head to tail) multimers.