Structural characteristics of the chloroplast <Emphasis Type="Italic">rpS16</Emphasis> intron in <Emphasis Type="Italic">Allium sativum</Emphasis> and related <Emphasis Type="Italic">Allium</Emphasis> species

The intron sequence of chloroplast rpS16 and the secondary structure of its pre-mRNA were characterized for the first time in 26 Allium sativum accessions of different ecologo-geographical origins and seven related Allium species. The boundaries and main stem-loop consensus sequences were identified for all six domains of the intron. Polymorphism was estimated for the total intron and its regions. The structural regions of the rpS16 intron proved to be heterogeneous for mutation rate and spectrum. Mutations were most abundant in domains II and IV, and transition predominated in domains I, III, V, and VI. In addition to structural elements and motifs typical for group IIB introns, several Allium-specific micro- and macrostructural mutations were revealed. A 290-bp deletion involving domains III and IV and part of domain V was observed in A. altaicum, A. fistulosum, and A. schoenoprasum. Several indels and nucleotide substitutions were found to cause a deviation of the pre-mRNA secondary structure from the consensus model of group II introns.     

Structure and polymorphism of a fragment of the Pain-1 vacuolar invertase locus in Solanum species

A fragment of the Pain-1 vacuolar invertase locus was sequenced and its structure and polymorphism were characterized in 17 species of the genus Solanum, including the subgenera Potatoe, Solanum, Leptostemonum, Minon, and Brevantherum. The fragment size varied from 603 to 977 bp as a result of multiple indels in the region of intron III. A total of 80 single nucleotide polymorphisms were found in the coding region, of which 34 caused amino acid substitution in the protein product. Several substitutions and indels were specific to individual taxons or taxon groups, including potato and tomato species. The genetic distances and phylogenetic trees obtained supported the commonly accepted taxonomic classification of the species, indicating that the Pain-1 fragment is suitable for taxonomic identification and phylogenetic studies in Solanaceae.     

Analysis of chloroplast <Emphasis Type="Italic">rpS16</Emphasis> intron sequences in Lemnaceae

Chloroplast rpS16 gene intron sequences were determined and characterized for twenty-five Lemnaceae accessions representing nine duckweed species. For each Lemnaceae species nucleotide substitutions and for Lemna minor, Lemna aequinoctialis, Wolffia arrhiza different indels were detected. Most of indels were found for Wolffia arrhiza and Lemna aequinoctialis. The analyses of intraspecific polymorphism resulted in identification of several gaplotypes in Lemna gibba and Lemna trisulca. Lemnaceae phylogenetic relationship based on rpS16 intron variability data has revealed significant differences between Lemna aequinoctialis and other Lemna species. Genetic distance values corroborated competence of Landoltia punctata separations from Spirodela into an independent generic taxon. The acceptability of rpS16 intron sequences for phylogenetic studies in Lemnaceae was shown.     

Polymorphism of sequences and the secondary structure of b/c intron of mitochondrial gene <Emphasis Type="Italic">nad1</Emphasis> in <Emphasis Type="Italic">Monotropa hypopitys</Emphasis> and related ericaceae species

Nucleotide sequences of b/c intron of the mitochondrial gene nad1 were determined for the first time and analyzed in 24 plants of Monotropa hypopitys and in three related Ericaceae species. Two mitotypes of b/c intron of the nad1 gene, differing in sequence lengths and the presence of a number of single nucleotide substitutions and indels, were revealed in M. hypopitys. For the first time, a possible pre-mRNA secondary structure of M. hypopitys b/c intron was determined and IBS/EBS binding sites and the borders of six functional domains were identified.     

茄科植物叶绿体基因组插入、缺失和核苷酸替代的发生方式及影响

摘要: 核苷酸替代和indels(插入、缺失统称)发生是进化的重要动力。以茄科植物为研究对象, 探讨茄属中番茄和马铃薯、烟草属中绒毛状烟草和普通烟草分化时叶绿体基因组indels和核苷酸替代的发生方式, 以及这两种突变对基因组造成的影响。结果显示: indels和核苷酸替代的发生都不是随意的。indels发生在A+T丰富的区域, 1 bp indels占据总数的30%以上, 大部分indels都为低于10 bp的较短片段。核苷酸替代表现出Ts(转换)/Tv(颠换)偏差, 但T→G, A→C颠换频率却明显增加。Ts/Tv比值出现种属特异性, 番茄和马铃薯比较时替代的Ts/Tv比值低于绒毛状烟草和普通烟草比较时Ts/Tv比值。不同物种替代的(A+T)/(G+C)比值有一定差异, 从而影响基因组的(G+C)%, 此比值的差异与形成物种的生长习性有一定的关系。     

Human ribosomal protein S13 inhibits splicing of its own pre-mRNA

Recombinant human ribosomal protein (rp) S13 was shown to specifically bind with its own pre-mRNA fragment containing the first exon, first intron, second exon, and a part of the second intron and to inhibit its splicing in vitro. The binding of rpS13 was specific: recombinant human rpS10 and rpS16 bound with the fragment to a lower extent. Moreover, rpS13 binding with the rpS13 pre-mRNA fragment was inhibited by non-labeled poly(AU) and an adenovirus pre-mRNA fragment to a lower extent than by the nonlabeled rpS13 pre-mRNA fragment. The specificity of splicing inhibition was inferred from the finding that, in contrast to rpS13, recombinant rpS10 and rpS16 did not affect the efficiency of first intron excision from the rpS13 pre-mRNA fragment. Enzymatic footprinting was used to determine the rpS13 pre-mRNA nucleotides whose accessibility to RNases T1, T2, and V1 changed in the presence of rpS13. Such nucleotides were detected close to the 5′ and 3′ splicing sites of the first intron. Analysis with the EMBOS-Align program showed that the nucleotide sequence of the first intron of the mammalian rpS13 pre-mRNA is conserved to a greater extent as compared with the other introns. It was assumed that the first intron plays an important role in regulating the expression of the rpS13 gene at the splicing level in all mammals.     

Complete chloroplast genome sequences from Korean ginseng (Panax schinseng Nees) and comparative analysis of sequence evolution among 17 vascular plants.

The nucleotide sequence of Korean ginseng (Panax schinseng Nees) chloroplast genome has been completed (AY582139). The circular double-stranded DNA, which consists of 156,318 bp, contains a pair of inverted repeat regions (IRa and IRb) with 26,071 bp each, which are separated by small and large single copy regions of 86,106 bp and 18,070 bp, respectively. The inverted repeat region is further extended into a large single copy region which includes the 5' parts of the rpsl9 gene. Four short inversions associated with short palindromic sequences that form stem-loop structures were also observed in the chloroplast genome of P. schinseng compared to that of Nicotiana tabacum. The genome content and the relative positions of 114 genes (75 peptide-encoding genes, 30 tRNA genes, 4 rRNA genes, and 5 conserved open reading frames [ycfs]), however, are identical with the chloroplast DNA of N. tabacum. Sixteen genes contain one intron while two genes have two introns. Of these introns, only one (trnL-UAA) belongs to the self-splicing group I; all remaining introns have the characteristics of six domains belonging to group II. Eighteen simple sequence repeats have been identified from the chloroplast genome of Korean ginseng. Several of these SSR loci show infra-specific variations. A detailed comparison of 17 known completed chloroplast genomes from the vascular plants allowed the identification of evolutionary modes of coding segments and intron sequences, as well as the evaluation of the phylogenetic utilities of chloroplast genes. Furthermore, through the detailed comparisons of several chloroplast genomes, evolutionary hotspots predominated by the inversion end points, indel mutation events, and high frequencies of base substitutions were identified. Large-sized indels were often associated with direct repeats at the end of the sequences facilitating intra-molecular recombination.     

Polymorphism and the secondary structure of the mitochondrial <Emphasis Type="Italic">nad1</Emphasis> gene b/c intron in <Emphasis Type="Italic">Malus</Emphasis> species and related Rosaceae species

Nucleotide sequences polymorphism of mitochondrial nad1 gene b/c intron was studied in 41 Malus accessions and 21 related Rosaceae accessions. The b/c intron sequence in genus Malus appeared to be very conservative, while in other studied Rosaceae species 126 variable sites and indels were detected in the intron sequence that varied in length from 1124 to 1456 bp. The predicted b/c intron pre-mRNA secondary structure for Malus species was determined; IBS/EBS binding sites and the boundaries of the six functional domains were identified.     

Nucleotide sequence variation of the chloroplast trnK/matK region in two wild Fagopyrum (Polygonaceae) species, F. leptopodum and F. statice

Nucleotide sequence polymorphisms of the intron of the chloroplast trnK (UUU) gene, including a matK gene, were investigated within two wild Fagopyrum species, F. leptopodum and F. statice, to assess the degree and pattern of the inter- and intraspecific differences in coding and noncoding chloroplast DNA regions in higher plants. Ten and five accessions were used for F. leptopodum and F. statice, respectively. The length of the trnK intron region in these species ranged from 2494 to 2506 bp. In the trnK intron, the net nucleotide substitution number per site (Da) between the two species was 0.00109, lower than the nucleotide diversity (pi), 0.00195 for F. leptopodum and 0.00144 for F. statice, suggesting a low level of interspecific divergence. This result seems to be due to the phylogenetic pattern that both species are interspersed with each other, which was revealed by the phylogenetic analyses based on the nucleotide substitutions and indels. In the matK gene region (1524 bp), seven and two nucleotide substitutions were found within F. leptopodum and F. statice, respectively. All of the nine nucleotide substitutions (eight of which were nonsynonymous) within and between F. leptopodum and F. statice were clustered in the 5' part of the matK gene region, and no variation was found in the 3' part. This suggests that most of the 3' part is occupied by the conserved domains that are important for the binding activity of the gene product to the precursor mRNA, and therefore implies that the 3' part is more functionally constrained than the 5' part.     

The discovery and phylogenetic implications of a novel 41 bp plastid DNA deletion in wild potatoes

Insertions and deletions (indels) are common in intergenic spacer regions of plastid DNA and can provide important phylogenetic characters for closely related species. For example, a 241-bp plastid DNA deletion in the trnV-UAC/ndhC intergenic spacer region has been shown to have major phylogenetic importance in determining the origin of the cultivated potato. As part of a phylogenetic study of the wild potato Solanum series Piurana group we screened 199 accessions of 38 wild potato species in nine of the 19 tuber-bearing (Solanum section Petota) series that have not been examined before for indels in the trnV-UAC/ndhC intergenic spacer region. A novel 41 bp deletion (but no 241 bp deletion) was discovered for 30 accessions of three species: S. chiquidenum (5 of 10 accessions), S. chomatophilum (19 of 28), and S. jalcae (6 of 6). Accessions with and without this deletion are found throughout much of the north-south range of all three species in northern and central Peru, but not east of the Marañón River. Multivariate morphological analyses of these 44 accessions showed no morphological associations to the deletion. The results suggest extensive interspecific gene flow among these three species, or a common evolutionary history among species that have never been suggested to be interrelated.     

Human ribosomal protein S16 inhibits excision of the first intron from its own pre-mRNA

Recombinant human ribosomal protein S16 (rpS16) is shown to bind specifically to a fragment of its own pre-mRNA that includes exons 1 and 2, intron 1, and part of intron 2, and to inhibit the splicing of the fragment in vitro. The weaker binding of other recombinant human ribosomal proteins, S10 and S13, to this pre-mRNA fragment indicated that the binding of rpS16 was specific. Besides, the poly(AU) and rpS16 mRNA fragment insignificantly affected the binding of rpS16 to its pre-mRNA, providing another evidence that the interaction was specific. rpS16 specifically inhibited the splicing of the pre-mRNA fragment, whereas recombinant rpS10 and rpS13 did not affect intron excision from this pre-mRNA fragment in contrast to rpS16. Those positions in rpS16 pre-mRNA fragment that were protected by rpS16 from cleavage by RNases T1, T2, and V1 were found to be located closely to the branch point and 3’ splice site in the pre-mRNA. The obtained results suggest the possibility of the autoregulation of rpS13 pre-mRNA splicing through the feedback mechanism.     

Hairpins involving both inverted and direct repeats are associated with homoplasious indels in non-coding chloroplast DNA of Taraxacum (Lactuceae: Asteraceae).

Sequence variation in 2.2 kb of non-coding regions of the chloroplast genome of eight dandelions (Taraxacum: Lactuceae) from Asia and Europe is interpreted in the light of the phylogenetic signal of base substitutions vs. indels (insertions-deletions). The four non-coding regions displayed a total of approximately 30 structural mutations of which 9 are potentially phylogenetically informative. Insertions, deletions, and an inversion were found that involved consecutive stretches of up to 172 bases. When compared to phylogenetic relationships of the chloroplast genomes based on nucleotide substitutions only, many homoplasious indels (33%) were detected that differed considerably in length and did not comprise simple sequence repeats typically associated with replication slippage. Though many indels in the intergenic spacers were associated with direct repeats, frequently, the variable stretches participated in inverted repeat stabilized hairpins. In each intergenic spacer or intron examined, nucleotide stretches ranging from 30 to 60 bp were able to fold into stabilized secondary structures. When these indels were homoplasious, they always ranked among the most stabilized hairpins in the non-coding regions. The association of higher order structures that involve both classes of repeats and parallel structural mutations in hot spot regions of the chloroplast genome can be used to differentiate among mutations that differ in phylogenetic reliability.     

Chloroplast DNA variation and genetic structure of Miscanthus sinensis in southwest China

Miscanthus sinensis is a dominant perennial C4 grass with the potential to being a feedstock crop in North America, Europe, and China. Variation in chloroplast DNA sequence was used to obtain information regarding the genetic diversity and structure of populations of M. sinensis in southwest China. Chloroplast DNA, trnL-F and rpl20-rps12 sequences from seventy-five individuals representing 14 populations of M. sinensis were used to study the sequence variation. Seven haplotypes and 16 polymorphic sites (2.7%) were identified. Five substitutions, 6 indels, and 5 existing substitutions and indels sites, were detected through splicing these two gene segments. The genetic diversity within the studied populations (diversity of haploids, h = 0.561, nucleotide diversity, π = 0.00504) was low, this may be affected by the relatively larger effect of genetic drift on the chloroplast DNA, reflecting smaller effective populations than nuclear DNA. Genetic variance within the populations was higher than that between the populations, suggesting that higher gene flow may exist within these populations. The results of parsimony network in seven haplotypes indicated that H1 and H2 may be ancient haplotypes, which may help guide future research on the origin of M. sinensis. Our results provide information on the genetic diversity and structure of M. sinensis and may assist future studies on the phylogeography of M. sinensis.     

Functional characterization of two novel splicing mutations of glucokinase gene associated with maturity-onset diabetes of the young type 2 (MODY2)

Two novel mutations in the glucokinase gene (GCK) have been identified in patients with maturity-onset diabetes of the young type-2 (MODY2), i.e., a C-for-G substitution at position ?1 of the acceptor splice site of intron 7 (c. 864-1G>C) and a synonymous c.666C>G substitution (GTC>GTG, p.V222V) at exon 6. An analysis of the splicing products obtained upon the transfection of human embryonic HEK293 cells with GCK minigene constructs carrying these mutations showed that both substitutions impaired normal splicing. As a result of c.864-1G>C, the usage of the normal acceptor site was blocked, which activated cryptic acceptor splice sites within intron 7 and generated several aberrant RNAs containing fragments of intron 7. The synonymous substitution c.666C>G created a novel donor splice site in exon 6, which results in the formation of an abnormal GCK mRNA with a 16-nucleotide deletion in exon 6. In vitro experiments on minigene splicing confirmed the inactivating effect of these mutations on glucokinase gene expression.     

Nucleotide sequence diversity at the methionine synthase locus in endangered Dunnia sinensis (Rubiaceae): an evaluation of the positive selection hypothesis

Methionine synthase is a key enzyme for the synthesis of the aspartate-derived methionine, the immediate precursor of S-adenosyl-methionine, which has been illustrated to be associated with plant growth and pathogen interactions. In this study we tested the positive selection hypothesis of molecular evolution of the methionine synthase gene in Dunnia sinensis. In the entire sample of 87 sequences, 22 haplotypes of introns and 16 haplotypes of exons were identified. An excess of polymorphism over the neutral expectation for the class of unique nucleotide polymorphisms was observed in both exon and intron sequences. Ten replacement substitutions versus six synonymous substitutions among lineages, although nonsignificant, revealed that some advantageous mutants might have been favored. The distribution of d(N)/d(S) > 1 at nodes between closely related haplotypes in the gene network also indicated weak and variable positive selection. Nevertheless, low levels of genetic diversity in exons (theta; = 0.0052) and introns (theta; = 0.0070) of the methionine synthase gene of the outcrossing Dunnia were also attributed to the endangered status of the species. The atpB-rbcL intergenic spacer of cpDNA and the ribosomal internal transcribed spacer of mtDNA were used to discern the relative effectiveness of natural selection from intrinsic evolutionary forces. The low levels of nucleotide polymorphisms in both organelle spacers and the significant population differentiation reflected the effect of population-species history and demography. Two major lineages of the methionine synthase gene genealogy were recovered corresponding to two geographic regions, a result that was consistent with organelle phylogenies. Both past fragmentation and recent habitat disturbance causing complete bottlenecks may have resulted in population decline and geographic isolation and may have led to the depletion of genetic variation at loci in nuclear and organelle genomes.     

Partial genomic organization of ribosomal protein S7 gene from malaria vector Anopheles stephensi

In this study, we describe the partial genomic organization of ribosomal protein S7 gene isolated from the mosquito Anopheles stephensi. Initially a 558 bp partial cDNA sequence was amplified as precursor mRNA sequence containing 223 bp long intron. 5＇ and 3＇ end sequences were recovered using end specific rapid amplification of cDNA ends （RACE） polymerase chain reaction. The full-length cDNA sequence was 914 nucleotide long with an open reading frame capable of encoding 192 amino acid long protein with calculated molecular mass of 22174 Da and a pI point of 9.94. Protein homology search revealed 〉75% identity to other insect＇s S7 ribosomal proteins. Analysis of sequence alignment revealed several highly conserved domains, one of which is related to nuclear localization signal （NLS） region of human rpS7. Interestingly, intron nucleotide sequence comparison with A. gambiae showed a lesser degree of conservation as compared to coding and untranslated regions. Like this, early studies on the genomic organization and cDNA/ Expressed sequence tag analysis （EST） could help in genome annotation ofA. stephensi, and would be likely to be sequenced in the future.     

Alignment and phylogenetic analysis of beta-fibrinogen intron 7 sequences among avian orders reveal conserved regions within the intron

We sequenced beta-fibrinogen intron 7 (beta-fibint 7) from 28 species of birds, representing 18 families in nine orders. Although the antiquity of the avian orders is estimated to be 55 to 90 Myr, and numerous indels have accrued among diverging lineages, the intron sequences were not difficult to align. However, alignment of avian sequences with mammal or snake sequences was difficult, and the residual phylogenetic signal was weak. beta-fibint 7 is an AT-rich intron, and its base composition varies little over the diversity of birds represented by our sample. Alignment of these anciently diverged sequences reveals at least five clusters of conserved nucleotides; at least two clusters appear to be in excess of the minimal set usually associated with intron excision, but their functions are unknown. Two equally most-parsimonious (MP) trees were found when indels were not included in the phylogenetic analysis, and six such trees were found when indels were included. The Neighbor-Joining and maximum-likelihood trees were identical to each other and to one of the MP trees in each MP analysis. Indels, as well as nucleotide substitutions, are phylogenetically informative, and bootstrap support exceeded 90% for 21 of 24 inferred nodes when indels were included in the MP analysis. All traditional orders represented by two or more species appear monophyletic. Relationships among avian orders are strongly supported with the exception of an inferred sister-group relationship between Caprimulgiformes and Columbiformes. A relatively close relationship between Piciformes and Passeriformes is inferred, at odds with earlier DNA-DNA hybridization studies but consistent with traditional classifications. Among Passeriformes, the traditional perspective of a sister-group relationship of suboscines and oscines is supported, as is the subsequent split of the oscines into a lineage representative of the Corvida before the diversification of the Passerida. The four species of owls divide into two strongly supported clades, corresponding to the widely accepted bifurcation of owls into two families, Tytonidae and Strigidae. A sister-group relationship between gallinaceous birds and waterfowl, the Galloanserae, is also strongly supported.     

Phylogenetic relationships among <Emphasis Type="Italic">Fagopyrum</Emphasis> species based on analysis of the <Emphasis Type="Italic">nad1</Emphasis> Gene b/c intron

For the first time, the b/c intron of the nad1 gene was characterized in 13 species of Fagopyrum and the applicability of the intron was demonstrated for identification of buckwheat species. The length of the b/c intron of the nad1 gene varied from 1217 bp in F. tataricum and F. cymosum to 1239 bp in F. capillatum. Species-specific SNPs and indels were identified. The genus phylogeny that was determined with the molecular approach correlated with phylogenetic data that are based on morphological characteristics. The NJ and MP dendrograms revealed distinct division of the analyzed species into two main groups, cymosum and urophyllum. The species of the urophyllum group were characterized by the presence of several synapomorphic substitutions and indels, including the insertion of 11 nucleotides, which is flanked by direct repeats.