A catalogue of splice junction and putative branch point sequences from plant introns.

Splice junction and possible branch point sequences have been collected from 177 plant introns. Consensus sequences for the 5' and 3' splice junctions and for possible branch points have been derived. The splice junction consensus sequences were virtually identical to those of animal introns except that the polypyrimidine stretch at the 3' splice junction was less pronounced in the plant introns. A search for possible branch points with sequences related to the yeast, vertebrate and fungal consensus sequences revealed a similar sequence in plant introns.     

SMARCA1基因组结构鉴定及其在Smith-Fineman-Myers综合征家系中的突变分析

为探讨SMARCA1基因在中国山东SFMS家系患者发生中的作用,采用计算机杂交结合DNA序列分析方法,首先确定了SMARCA1基因的基因组结构,发现该基因的基因组DNA全长超过71．7kb,含有24个外显子和23个内含子,所有外显子和内含子接头皆遵循GT-AG法则,基因组结构的阐明,为进行基因突变检测和分析其生物学功能奠定了基础。在以上分析的基础上,通过PCR扩增结合测序分析,对在山东省发现的1个SFMS家系患者的SMARCA1基因的全部外显子和外显子内含子接头序列进行了基因突变检测,未检测到导致疾病的突变,提示中国山东SFMS家系患者不是由于SMARCA1基因编码区域内基因突变所致。     

Structure of the feline c-fes/fps proto-oncogene: genesis of a retroviral oncogene.

The nucleotide sequence of the feline c-fes/fps proto-oncogene was analyzed. Comparison with v-fes and v-fps revealed that all v-fes/fps homologous sequences were dispersed over 11 kilobase pairs in 19 interspersed segments. All segments, numbered exon 1 to exon 19 as in the chicken and human loci, were flanked by consensus splice junctions. The putative promoter region contained a CATT sequence and three CCGCCC motifs which were also found in the human locus at similar positions. About 200 nucleotides downstream of a translational stop codon in exon 19, a putative poly(A) addition signal was identified. Using the putative translation initiation codon in exon 2, a 93,000-molecular-weight protein could be deduced. This protein resembled very well the putative protein of the human c-fes/fps proto-oncogene (94% overall homology) and, although less well, the putative protein of the chicken c-fes/fps proto-oncogene (70% overall homology). As far as the feline c-fes/fps proto-oncogene sequences transduced to the Gardner-Arnstein (GA) and Snyder-Theilen (ST) strains of feline sarcoma virus (FeSV) are concerned, homology in deduced amino acid sequences between the GA- and ST-v-fes viral oncogenes and the proto-oncogene was 99%. Analysis of the recombination junctions between feline leukemia virus and v-fes sequences in GA- and ST-FeSV proviral DNA revealed for the left-hand junction the involvement of homologous recombination, presumably at the DNA level. The right-hand junction, which appeared identical in the GA-FeSV and ST-FeSV genomes, could have been the result of a site-specific recombination at the RNA level.     

Nine introns with conserved boundary sequences in the Euglena gracilis chloroplast ribulose-1,5-bisphosphate carboxylase gene

The single, chloroplast encoded gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) from Euglena gracilis is found to contain nine intervening sequences. The intervening sequences were identified by heteroduplex analysis between Euglena rbcL and the non-intron-containing rbcL from Spinacea oleracea, by electron microscopy of Euglena rbcL DNA-mRNA hybrids, and by cloning, restriction endonuclease analysis, and partial DNA sequencing. The identification, locus, and coding properties for six of ten exons was confirmed by partial DNA sequence analysis. Each of the nine introns in the approximately 6.5 kb rbcL locus is approximately 0.5 kb in length. The DNA sequence of five 3'-intron/5'-exon and four 3'-exon/5'-intron boundaries are highly conserved. A proposed consensus sequence is (formula; see text) These conserved sequences could play a role in an mRNA splicing mechanism in chloroplasts analogous to that in eucaryotic nuclei.     

The RuvC protein dimer resolves Holliday junctions by a dual incision mechanism that involves base-specific contacts.

The Escherichia coli RuvC protein resolves DNA intermediates produced during genetic recombination. In vitro, RuvC binds specifically to Holliday junctions and resolves them by the introduction of nicks into two strands of like polarity. In contrast to junction recognition, which occurs without regard for DNA sequence, resolution occurs preferentially at sequences that exhibit the consensus 5'-(A/T)TT/(G/C)-3' (where / indicates the site of incision). Synthetic Holliday junctions containing modified cleavage sequences have been used to investigate the mechanism of cleavage. The results indicate that specific DNA sequences are required for the correct docking of DNA into the two active sites of the RuvC dimer. In addition, using chemically modified oligonucleotides to introduce a hydrolysis-resistant 3'-S-phosphorothiolate linkage at the cleavage site, it was found that, as long as the sequence requirements are fulfilled, the two incisions could be uncoupled from each other. These results indicate that RuvC protein resolves Holliday junctions by a mechanism similar to that exhibited by certain restriction enzymes.     

Terminal structure and heterogeneity in human cytomegalovirus strain AD169.

We have characterized the heterogeneity occurring at the junction of the long (L) and short (S) segments and at the termini of the strain AD169 human cytomegalovirus (HCMV) genome by restriction endonuclease mapping and nucleotide sequence analyses. The HCMV a sequence was identified by its position at both termini and inverted orientation at the L-S junction. Heterogeneity at both termini and the L-S junction was generated by the presence of fused and tandem a sequences. Some S termini lacked an a sequence. In addition, near the L terminus and at the L-S junction there were a variable number of 217-base-pair (bp) XhoI fragments arranged in tandem. The 217-bp fragments consisted of a portion of the a and adjacent b sequences (in the L-segment repeat) bounded by the same direct repeats (DR1) found at the boundaries of the a sequence. A model for the generation of these heterogeneous fragments is presented. We also determined the sequence of seven cloned terminal fragments, five from the L terminus and two from the S terminus. All L termini contained identical terminal sequences ending with base 32 of a 33-bp DR1. The S termini differed from each other and from the L-segment termini. One S terminus lacked an a sequence and terminated within S-segment repeat (c) sequences. The second S terminus contained an a sequence and terminated with bases 20 to 33 of a 33-bp DR1. A comparison of the cloned L and S terminal sequences with cloned L-S junction sequences suggested that the termini contained 3' single base extensions which were removed during the cloning. We also show that the herpesvirus conserved sequence is in a similar position relative to the termini of HCMV and several other herpesviruses, thus adding further support for the role of the sequence in the maturation of viral DNA.     

Identification of human telomeric repeat motifs at the genome termini of human herpesvirus 7: structural analysis and heterogeneity.

Human herpesvirus 6 (HHV-6) and HHV-7 are closely related T-lymphotropic betaherpesviruses which share a common genomic organization and are composed of a single unique component (U) that is bounded by direct repeats (DRL and DRR). In HHV-6, a sequences have been identified at each end of the DR motifs, resulting in the arrangement aDRLa-U-aDRRa. In order to determine whether determine whether HHV-7 contains similar a sequences, we have sequenced the DRL-U and U-DRR junctions of HHV-7 strain JI, together with the DRR.DRL junction from the head-to-tail concatamer that is generated during productive virus infection. In addition, we have sequenced the genomic termini of an independent isolate of HHV-7. As in HHV-6, a (GGGTTA)n motif identical to the human telomeric repeat sequence (TRS) was identified adjacent to, but not at, the genome termini of HHV-7. The left genome terminus and the U-DRR junction contained a homolog of the consensus herpesvirus packaging signal, pac-1, followed by short tandem arrays of TRSs separated by single copies of a second 6-bp repeat. This organization is similar to the arrangement found at U-DRR in HHV-6 but differs from it in that the TRS arrays are considerably shorter in HHV-7. The right genome terminus and the DRL-U junction contained a homolog of the consensus herpesvirus packaging signal, pac-2, followed by longer tandem arrays of TRSs separated by single copies of either a 6-bp or a 14-bp repeat. This arrangement is considerably more complex than the simple tandem array of TRSs that is present at the corresponding genomic location in HHV-6 and corresponds to a site of both inter- and intrastrain heterogeneity in HHV-7. The presence of TRSs in lymphotropic herpesviruses from humans (HHV-6 and HHV-7), horse (equine herpesvirus 2), and birds (Marek's disease virus) is striking and suggests that these sequences may have functional or structural significance.     

Uncommon Alu-mediated NF1 microdeletion with a breakpoint inside the NF1 gene

Neurofibromatosis type 1 (NF1) microdeletion syndrome is caused by haploinsufficiency of the NF1 gene and of gene(s) located in adjacent flanking regions. Most of the NF1 deletions originate by nonallelic homologous recombination between repeated sequences (REP-P and -M) mapped to 17q11.2, while a few uncommon deletions show unusual breakpoints. We characterized an uncommon 1.5-Mb deletion of an NF1 patient displaying a mild phenotype. We applied high-resolution FISH analysis allowing us to obtain the sequence of the first junction fragment of an uncommon deletion showing the telomeric breakpoint inside the IVS23a of the NF1 gene. Sequence analysis of the centromeric and telomeric boundaries revealed that the breakpoints were present in the AluJb and AluSx regions, respectively, showing 85% homology. The centromeric breakpoint is localized inside a chi-like element; a few copies of this sequence are also located very close to both breakpoints. The in silico analysis of the breakpoint intervals, aimed at identifying consensus sequences of several motifs usually involved in deletions and translocations, suggests that Alu sequences, probably associated with the chi-like element, might be the only recombinogenic motif directly mediating this large deletion.     

Functional Identification and Analysis of cis-Acting Sequences Which Mediate Genome Cleavage and Packaging in Human Herpesvirus 6

Sequences present at the genomic termini of herpesviruses become linked during lytic-phase replication and provide the substrate for cleavage and packaging of unit length viral genomes. We have previously shown that homologs of the consensus herpesvirus cleavage-packaging signals, pac1 and pac2, are located at the left and right genomic termini of human herpesvirus 6 (HHV-6), respectively. Immediately adjacent to these elements are two distinct arrays of human telomeric repeat sequences (TRS). We now show that the unique sequence element formed at the junction of HHV-6B genome concatemers (pac2-pac1) is necessary and sufficient for virally mediated cleavage of plasmid DNAs containing the HHV-6B lytic-phase origin of DNA replication (oriLyt). The concatemeric junction sequence also allowed for the packaging of these plasmid molecules into intracellular nucleocapsids as well as mature, infectious viral particles. In addition, this element significantly enhanced the replication efficiency of oriLyt-containing plasmids in virally infected cells. Experiments revealed that the concatemeric junction sequence possesses an unusual, S1 nuclease-sensitive conformation (anisomorphic DNA), which might play a role in this apparent enhancement of DNA replication—although additional studies will be required to test this hypothesis. Finally, we also analyzed whether the presence of flanking viral TRS had any effect on the functional activity of the minimal concatemeric junction (pac2-pac1). These experiments revealed that the TRS motifs, either alone or in combination, had no effect on the efficiency of virally mediated DNA replication or DNA cleavage. Taken together, these data show that the cleavage and packaging of HHV-6 DNA are mediated by cis-acting consensus sequences similar to those found in other herpesviruses, and that these sequences also influence the efficiency of HHV-6 DNA replication. Since the adjacent TRS do not influence either viral cleavage and packaging or viral DNA replication, their function remains uncertain.     

Characterization of recombinant hepatitis A virus genomes containing exogenous sequences at the 2A/2B junction

Hepatitis A virus (HAV) differs from other members of the family Picornaviridae in that the cleavage of the polyprotein at the 2A/2B junction, commonly considered to be the primary polyprotein cleavage by analogy with other picornaviruses, is mediated by 3C(pro), the only proteinase encoded by the virus. However, it has never been formally demonstrated that the 2A/2B junction is the site of primary cleavage, and the actual function of the 2A sequence, which lacks homology with sequence of other picornaviruses, remains unknown. To determine whether 2A functions in cis as a precursor with the nonstructural proteins, we constructed dicistronic HAV genomes in which a heterologous picornaviral internal ribosome entry site was inserted at the 2A/2B junction. Transfection of permissive FRhK-4 cells with these dicistronic RNAs failed to result in the rescue of infectious virus, indicating a possible cis replication function spanning the 2A/2B junction. However, infectious virus was recovered from recombinant HAV genomes containing exogenous protein-coding sequences inserted in-frame at the 2A/2B junction and flanked by consensus 3C(pro) cleavage sites. The replication of these recombinants was less efficient than that of the parent virus but was variable and not dependent upon the length of the inserted sequence. An HAV recombinant containing a 420-nt insertion encoding the bleomycin resistance protein Zeo was stable for up to five passages in cell culture. Inserted sequences were deleted from replicating viruses, but this did not result from homologous recombination at the flanking 3C(pro) cleavage sites, since the 5' and 3' segments of the inserted sequence were retained in the deletion mutants. These results indicate that the HAV polyprotein can tolerate an insertion at the 2A/2B junction and that the 2A polypeptide does not function in cis as a 2AB precursor. Recombinant HAV genomes containing foreign protein-coding sequences inserted at the 2A/2B junction are novel and potentially useful protein expression vectors.     

Exonic splicing enhancer motif recognized by human SC35 under splicing conditions

Exonic splicing enhancers (ESEs) are important cis elements required for exon inclusion. Using an in vitro functional selection and amplification procedure, we have identified a novel ESE motif recognized by the human SR protein SC35 under splicing conditions. The selected sequences are functional and specific: they promote splicing in nuclear extract or in S100 extract complemented by SC35 but not by SF2/ASF. They can also function in a different exonic context from the one used for the selection procedure. The selected sequences share one or two close matches to a short and highly degenerate octamer consensus, GRYYcSYR. A score matrix was generated from the selected sequences according to the nucleotide frequency at each position of their best match to the consensus motif. The SC35 score matrix, along with our previously reported SF2/ASF score matrix, was used to search the sequences of two well-characterized splicing substrates derived from the mouse immunoglobulin M (IgM) and human immunodeficiency virus tat genes. Multiple SC35 high-score motifs, but only two widely separated SF2/ASF motifs, were found in the IgM C4 exon, which can be spliced in S100 extract complemented by SC35. In contrast, multiple high-score motifs for both SF2/ASF and SC35 were found in a variant of the Tat T3 exon (lacking an SC35-specific silencer) whose splicing can be complemented by either SF2/ASF or SC35. The motif score matrix can help locate SC35-specific enhancers in natural exon sequences.     

Isolation of different types of birnavirus from ayu Plecoglossus altivelis and amago salmon Oncorhynchus rhodurus cultured in the same geographic area

A birnavirus was recently isolated from cultured ayu Plecoglossus altivelis on Shikoku island, Japan. The diseased fish displayed vertebral or vertical curvature and mild haemorrhage around the brain. Cytopathic effects (CPE) of the virus, including cell roundness, filamentous change and cell lysis, were observed in CHSE-214, RTG-2 and RSBK-2 cells. The virus isolated from ayu, designated the AY-98 strain, was found to be antigenically related to the marine birnavirus (MABV) Y-6 strain that originated from yellowtail Seriola quinqueradiata. AY-98 had a bi-segmented RNA genome and the same nucleotide sequence in the 310 bp VP2/NS junction as MABV Y-6. At the same time that the ayu epizootics occurred, another birnavirus (AM-98) was isolated from amago salmon Oncorhynchus rhodurus which were cultured 66 km away from the ayu farm. AM-98 showed a similar CPE and had the same host cell ranges as AY-98. However, AM-98 was serologically similar to the VR-299 strain of infectious pancreatic necrosis virus (IPNV) and their nucleotide sequences in the VP2/NS junction region showed 98% homology without changes at the amino acid level. In this study, the ayu strain AY-98 was grouped into MABV, whereas the amago salmon strain AM-98 was grouped into IPNV. This indicates that the 2 birnaviruses originated from different sources in spite of the fact that the places where they were isolated are close to one another. The results in this paper show a new aspect of the traditional consensus that the same serogroup of birnavirus distribute in close geographic areas.     

Identification of preferred actinomycin-DNA binding sites by the combinatorial method REPSA.

An important question in the study of ligand-DNA interactions is the determination of binding specificity. Here, we used the combinatorial method restriction endonuclease protection, selection, and amplification (REPSA) to identify the preferred duplex DNA-binding sites of the antineoplastic agent actinomycin D. After 10 rounds of REPSA, over 95% of the cloned DNAs exhibited significantly reduced FokI restriction endonuclease cleavage in the presence of 1 microM actinomycin. A chi(2) statistical analysis of their sequences found that 39 of the 45 clones contained one or more copies of the sequence 5'-(T/A)GC(A/T)-3', giving a p<0.001 for this consensus. A DNase I footprinting analysis of the cloned DNAs found that all possessed relatively high affinity actinomycin-binding sites with apparent dissociation constants between 12 and 258nM (average 98nM). The average footprint encompassed 7.6 bases and in most cases (90%) included one or more consensus sequences. Interestingly, several of the selected clones contained overlapping consensus sequences (e.g., 5'-TGCTGCT-3'), suggesting that such close proximity DNA-binding sites may actually be preferred by actinomycin under physiological conditions.     

Repeated regions in mitochondrial genomes: Distribution, origin and evolutionary significance

All complete or nearly complete mitochondrial genomes of Metazoa (2819) have been subject to bioinformatic analysis to investigate the distribution and features of repeated and palindromic sequences. Repeats are ubiquitous, with 29.9% of genomes containing at least one and 1.95% of total genome length being repeated. Repeat boundaries were tested for the presence of secondary structure motifs, consensus sequences or small repeats, features generally reported as associated with duplications. No significant relationship was detected, suggesting the non ubiquitousness of such features. A mechanism related to gene conversion is proposed to explain the origin of small interspersed repeats.