Organization and evolutionary progress of a dispersed repetitive family of sequences in widely separated rodent genomes

The genomes of Mus musculus and other rodent species share a long conserved family of sequences that are dispersed and abundant (approx. 20,000 copies), and that have several novel features of organization and evolution. EcoR1 restriction of M. musculus DNA reveals a prominent 1350 bp² set of sequences. Two nonhomologous sequences of 850 and 500 bp, representing almost the total population of the 1350 bp repeats, were used to examine the detailed organization of the dispersed family and its surrounding sequences using a combination of restriction analysis and “Southern” hybridization. The 1350 bp sequence is contained within a longer repeating unit of approximately 3 kb that is dispersed amongst a wide variety of non-homologous and seemingly non-repetitive sequences. At some sites within the 3 kb repeat, considerable sequence heterogeneity has been found between members of the family, such that the family can be divided into largely non-overlapping subsets (or “segments”) according to the positioning of HinIII sites. Underlying the segmental organization there is a low background overlap of each segment with every other. Some but not all members of the family and its variants have been located on the X-chromosome in a Chinese hamster, M. musculus, X chromosome cell line: suggesting a wide genomic dispersion of the family. Homologous repeated sequences to the M. musculus 1350 bp repeat have been identified in species of Mus and Apodemus, with strikingly similar features of organization and dispersion. In M. spretus a 1350 bp sequence is contained within a dispersed repeat of at least 2·9 kb. However, the majority of M. spretus repeats contain an additional restriction site not present in the equivalent M. musculus array, suggesting a mechanism of widespread substitution or “conversion” of one variant by another in each genome. Apodemus sylvaticus possesses two dispersed and homologous families of 1350 bp and 1850 bp repetition, respectively, which contain sequences that have diverged from M. musculus to differing extents. A. mystacinus possesses only one family of dispersed and homologous repeats of 1850 bp. The majority of members within each Apodemus homologous family also contain characteristic variant restriction-site arrangements. The mechanisms underlying the spread of such variants within each array; the generation of segmental patterns; and the evolutionary conservation of this mouse interspersed family (MIF-1) are discussed in relation to the present knowledge of the organization and activity of other dispersed sequence families.     

Identification ofTnr3, aSuppressor-Mutator/Enhancer-like transposable element from rice

We isolated members of the retroposon family p-SINE1 in rice and found that one member contained an insertion. A 3-bp sequence at the insertion site within p-SINE1 appeared duplicated. The insertion sequence, 1536 bp in length, carried imperfect inverted repeats of about 13 bp at its termini which begin with 5′-CACTA--- -3′; these repeats are similar to those found in members of theEn/Spm transposable element family. These results indicate that the insertion sequence is a transposable element belonging to theEn/Spm family and is thus namedTnr3 (transposable element inrice no.3). In fact,Tnr3 carried long subterminal regions containing direct and inverted repeats of short DNA sequences of 15 bp, another characteristic of theEN/Spm family. The subterminal repeat sequences inTnr3 are, however, of two kinds, although they share homology with each other.Tnr3 and its relatives were present in multiple copies in rice. Considering the length ofTnr3, it cannot represent an autonomous type element, but is a non-autonomous element probably derived by deletion from an autonomous transposon.     

Intron 1 mediated regulation of bovine prion protein gene expression: Role of donor splicing sites, sequences with potential enhancer and suppressor activities

Prion protein plays a key role in the pathogenesis of transmissible spongiform encephalopathies. Because changes in expression of the prion protein gene (PRNP) alter the incubation time and severity of prion diseases. Our previous work revealed a strong association between the promoter (spanning base pairs (bp) −88 to −30) and intron 1 (spanning bp +114 to +892) that leads to optimum expression of the bovine PRNP. Here, we employed two mutation analysis strategies (deletion and insertion) and two reporter assay systems (luciferase and GFP expression) to define the regulatory domains within intron 1 and further elucidate its role in regulating the promoter activity of the bovine prion protein gene. We identified DNA sequences with potential suppressor and enhancer activities within the 5′ end of intron 1. Moreover stability analyses for PRNP mRNAs demonstrated that splicing sites and mechanism are critical for bovine PRNP expression.     

Origin of the main class of repetitive DNA within selected Pennisetum species

In an attempt to identify relationships among genomes of the allotetraploid Pennisetum purpureum Schumach and closely related Pennisetum species with which it can be successfully hybridized, repetitive DNA sequences were examined. Digestion with KpnI revealed two highly repetitive fragments of 140 by and 160 bp. The possibility that these sequences could be used as genome markers was investigated. Average sequences were determined for the 140 by and 160 by KpnI families from P. purpureum and P. squamulatum Fresen. Average sequences (based upon four or five repeats) were determined for the P. glaucum (L.) R. Br. 140 by KpnI family and the diploid P. hohenackeri Hochst. ex Steud. 160 bp KpnI family. The average sequences of the 160 by KpnI families in P. purpureum and P. squamulatum differ by only nine bases. The 140 by KpnI families of the three related species, P. purpureum, P. squamulantum, and P. glaucum are nearly identical, and thus likely represent a recent divergence from a common progenitor or a common genome. Each repetitive sequence may contain internal duplications, which probably diverged following amplification of the original sequence. The 140 by KpnI repeat probably evolved from the 160 by KpnI repeat since the missing 18 by segment is part of the internal duplication that is otherwise conserved in the subrepeats. Tandemly arrayed repetitive sequences in plants are likely to be composed of subrepeats which have been duplicated and amplified.     

Replication pattern of human repeated DNA sequences

Either aphidicolin- or thymidine-synchronized human HL-60 cells were used to study the replication pattern of a family of human repetitive DNA sequences, the EcoRI 340 bp family (αRI-DNA), and of the ladders of fragments generated in total human DNA after digestion with XbaI and HaeIII (alpha satellite sequences). DNAs replicated in early, middle-early, middle-late and late S periods were labelled with BUdR or with [³H]thymidine. The efficiency of the cell synchronization procedure was confirmed by the transition from a high-GC to a high-AT average base composition of the DNA synthesized going from early to late S periods. By hybridizing EcoRI 340 bp repetitive fragments to BUdR-DNAs it was found that this family of sequences is replicated throughout the entire S period. Comparing fluorograph densitometric scans of [³H]DNAs to the scans of ethidium bromide patterns of total HL-60 DNA digested with XbaI and HaeIII, it was observed that DNA synthesized in different S periods is characterized by approximately the same ladder of fragments, while the intensity of each band may vary through the S phase; in particular, the XbaI 2.4 kb fragment becomes undetectable in late S.     

Sub-terminal sequences modulating IS30 transposition in vivo and in vitro

Inverted repeats of insertion sequences (ISs) are indispensable for transposition. We demonstrate that sub-terminal sequences adjacent to the inverted repeats of IS30 are also required for optimal transposition activity. We have developed a cell-free recombination system and showed that the transposase catalyses formation of a figure-of-eight transposition intermediate, where a 2 bp long single strand bridge holds the inverted repeat sequences (IRs) together. This is the first demonstration of the figure-of-eight structure in a non-IS3 family element, suggesting that this mechanism is likely more widely adopted among IS families. We show that the absence of sub-terminal IS30 sequences negatively influences figure-of-eight production both in vivo and in vitro. These regions enhance IR-IR junction formation and IR-targeting events in vivo. Enhancer elements have been identified within 51 bp internal to IRL and 17 bp internal to IRR. In the right end, a decanucleotide, 5′-GAGATAATTG-3′, is responsible for wild-type activity, while in the left end, a complex assembly of repetitive elements is required. Functioning of the 10 bp element in the right end is position-dependent and the repetitive elements in the left end act cooperatively and may influence bendability of the end. In vitro kinetic experiments suggest that the sub-terminal enhancers may, at least partly, be transposase-dependent. Such enhancers may reflect a subtle regulatory mechanism for IS30 transposition.     

Families of restriction enzymes: an analysis prompted by molecular and genetic data for type ID restriction and modification systems

Current genetic and molecular evidence places all the known type I restriction and modification systems of Escherichia coli and Salmonella enterica into one of four discrete families: type IA, IB, IC or ID. StySBLI is the founder member of the ID family. Similarities of coding sequences have identified restriction systems in E.coli and Klebsiella pneumoniae as probable members of the type ID family. We present complementation tests that confirm the allocation of EcoR9I and KpnAI to the ID family. An alignment of the amino acid sequences of the HsdS subunits of StySBLI and EcoR9I identify two variable regions, each predicted to be a target recognition domain (TRD). Consistent with two TRDs, StySBLI was shown to recognise a bipartite target sequence, but one in which the adenine residues that are the substrates for methylation are separated by only 6 bp. Implications of family relationships are discussed and evidence is presented that extends the family affiliations identified in enteric bacteria to a wide range of other genera.     

Complete Genome Sequences of Elephant Endotheliotropic Herpesviruses 1A and 1B Determined Directly from Fatal Cases

A highly lethal hemorrhagic disease associated with infection by elephant endotheliotropic herpesvirus (EEHV) poses a severe threat to Asian elephant husbandry. We have used high-throughput methods to sequence the genomes of the two genotypes that are involved in most fatalities, namely, EEHV1A and EEHV1B (species Elephantid herpesvirus 1, genus Proboscivirus, subfamily Betaherpesvirinae, family Herpesviridae). The sequences were determined from postmortem tissue samples, despite the data containing tiny proportions of viral reads among reads from a host for which the genome sequence was not available. The EEHV1A genome is 180,421 bp in size and consists of a unique sequence (174,601 bp) flanked by a terminal direct repeat (2,910 bp). The genome contains 116 predicted protein-coding genes, of which six are fragmented, and seven paralogous gene families are present. The EEHV1B genome is very similar to that of EEHV1A in structure, size, and gene layout. Half of the EEHV1A genes lack orthologs in other members of subfamily Betaherpesvirinae, such as human cytomegalovirus (genus Cytomegalovirus) and human herpesvirus 6A (genus Roseolovirus). Notable among these are 23 genes encoding type 3 membrane proteins containing seven transmembrane domains (the 7TM family) and seven genes encoding related type 2 membrane proteins (the EE50 family). The EE50 family appears to be under intense evolutionary selection, as it is highly diverged between the two genotypes, exhibits evidence of sequence duplications or deletions, and contains several fragmented genes. The availability of the genome sequences will facilitate future research on the epidemiology, pathogenesis, diagnosis, and treatment of EEHV-associated disease.     

Study on Sequences of Ribosomal DNA Internal Transcribed Spacers of Clams Belonging to the Veneridae Family (Mollusca: Bivalvia)

The first and second internal transcribed spacer (ITS1 and ITS2) regions of the ribosomal DNA from four species, Meretrix meretrix L., Cyclina sinensis G., Mercenaria mercenaria L., and Protothaca jedoensis L., belonging to the family Veneridae were amplified by PCR and sequenced. The size of the ITS1 PCR amplification product ranged from 663 bp to 978 bp, with GC contents ranging from 60.78% to 64.97%. The size of the ITS1 sequence ranged from 585 bp to 900 bp, which is the largest range reported thus far in bivalve species, with GC contents ranging from 61.03% to 65.62%. The size of the ITS2 PCR amplification product ranged from 513 bp to 644 bp, with GC contents ranging from 61.29% to 62.73%. The size of the ITS2 sequence ranged from 281 bp to 412 bp, with GC contents ranging from 65.21% to 67.87%. Extensive sequence variation and obvious length polymorphisms were noted for both regions in these species, and sequence similarity of ITS2 was higher than that of ITS1 across species. The complete sequences of 5.8S ribosomal RNA gene were obtained by assembling ITS1 and ITS2 sequences, and the sequence length in all species was 157 bp. The phylogenetic tree of Veneridae clams was reconstructed using ITS2-containing partial sequences of both 5.8S and 28S ribosomal DNA as markers and the corresponding sequence information in Arctica islandica as the outgroup. Tree topologies indicated that P. jedoensis shared a close relationship with M. mercenaria and C. sinensis, a distant relationship with other species.     

Mitochondrial DNA as effective molecular markers for the genetic variation and phylogeny of the family Osteoglossidae

The present study examined the genetic variation of the family Osteoglossidae from different geographical locations based on the mitochondrial NADH dehydrogenase subunit 2 (ND2) and ATPase subunit 6 (ATPase6) genes; we then re-constructed the phylogenetic relationships using the two sequences in combination. The results showed that the partial sequences of mitochondrial ND2 and ATPase6 of the family Osteoglossidae were 813 bp and 669 bp, respectively. A total of 42 species-specific nucleotide positions of the family Osteoglossidae were found to be useful for molecular identification. The sequence variation showed greater differences (8.3% ~ 28.1% for the combined sequences, 8.3% ~ 26.7% for the ND2 gene, and 9.3% ~ 28.7% for the ATPase6 gene) among the different species of Osteoglossidae, and there was a significant association between the genetic difference and geographical location. Phylogenetic analyses using neighbor-joining, Bayesian inference, and maximum parsimony (MP) methods based on the combined sequences of the two genes were able to distinguish the different species and were in agreement with the existing taxonomy based on morphological characters and in association with the geographical distribution among seven species of the family Osteoglossidae.     

Coordinate regulation of bovine prion protein gene promoter activity by two Sp1 binding site polymorphisms

Relationships between insertion/deletion (Ins/Del) polymorphisms of the bovine prion protein gene (PRNP) promoter and bovine spongiform encephalopathy (BSE) susceptibility have been reported. Our previous study has shown that polymorphisms of −6C → T included in the specific protein 1 (Sp1) site in the 5′-flanking region of bovine PRNP influence the promoter activity of bovine PRNP. The present study shows that 12 and 23 bp Ins/Del polymorphisms in the upstream region and an additional polymorphism (−47C → A) in the Sp1 binding site coordinately affect the promoter activity. Reporter gene assays demonstrated that the bovine PRNP promoter containing −47A and 23 bp Del/12 bp Ins or 23 bp Ins/12 bp Ins showed lower promoter activity compared with other haplotypes (23 bp Del/12 bp Ins or 23 bp Ins/12 bp Del with −47C) or the wild-type haplotype (23 bp Del/12 bp Del with −47C). Furthermore, gel shift assays showed that the binding activity of Sp1 to the PRNP promoter was influenced by both polymorphisms with corresponding effects on the promoter activity. The coordinate regulation of the bovine PRNP promoter suggests the two Sp1 binding site polymorphisms control Sp1 binding to the PRNP promoter and its activity.     

Cloning of the cDNAs coding for cat growth hormone and prolactin

Characterization of the prolactin (PRL) amino acid (aa) or cDNA sequences has not been reported for any member of the Felidae family. We cloned cat growth hormone (cGH) and cat PRL (cPRL) cDNA sequences from a feline pituitary cDNA library. High homology between species allowed bovine PRL (bPRL) and bGH cDNA clones to be used to identify clones encoding the 229-aa cPRL and 216-aa cGH sequences. The cGH protein is most homologous to pig and dog GH. Similarly, cPRL shares the most aa identity to pig PRL (pPRL). Northern blot analysis revealed the mRNA size for cGH and cPRL to be approx. 1 and 1.1 kb, respectively. These results reveal that GH and PRL from the Felidae family are highly conserved to other families of GH and PRL.     

The draft chromosome-level genome assembly of tetraploid ground cherry (Prunus fruticosa Pall.) from long reads

Cherries are stone fruits and belong to the economically important plant family of Rosaceae with worldwide cultivation of different species. The ground cherry, Prunus fruticosa Pall., is an ancestor of cultivated sour cherry, an important tetraploid cherry species. Here, we present a long read chromosome-level draft genome assembly and related plastid sequences using the Oxford Nanopore Technology PromethION platform and R10.3 pore type. We generated a final consensus genome sequence of 366 Mb comprising eight chromosomes. The N50 scaffold was ~44 Mb with the longest chromosome being 66.5 Mb. The chloroplast and mitochondrial genomes were 158,217 bp and 383,281 bp long, which is in accordance with previously published plastid sequences. This is the first report of the genome of ground cherry (P. fruticosa) sequenced by long read technology only. The datasets obtained from this study provide a foundation for future breeding, molecular and evolutionary analysis in Prunus studies.     

新基因水稻 OsLSD1 的克隆及拟南芥和水稻类 LSD1 基因家族的生物信息学分析

类 LSD1 (LSD1-like) 基因家族是一类特殊的 C2C2 型锌指蛋白基因,编码植物特有的转录因子 . 目前已经研究的 2 个成员拟南芥 LSD1 (lesions stimulating disease resistance 1) 和 LOL1 (LSD-One-Like 1) 基因均参与植物细胞程序化死亡 (programmed cell death, PCD) 的调控 . 从水稻 cDNA 文库中克隆到 1 个类 LSD1 基因,命名为 OsLSD1. 该基因长 988 bp ,包含一个 432 bp 的开放阅读框,推导的氨基酸序列 (143 个氨基酸 ) 含有 3 个内部保守的锌指结构域 . DNA 印迹结果表明 OsLSD1 基因在水稻基因组中为单拷贝,且在根、茎和叶中表达 . 借助于生物信息学分析技术,从拟南芥和水稻数据库中各识别出 5 个和 7 个 ( 包括 OsLSD1) 类 LSD1 基因 . 分析了这些类 LSD1 基因的结构,蛋白质结构域组成 . 系统进化分析表明,无论基于编码区的核苷酸或氨基酸序列都可以将这些类 LSD1 基因分为 2 类 . 虽然不存在拟南芥或水稻特有的类 LSD1 蛋白,但有些结构域是水稻所特有的,也有些基因是来源于复制事件 .