In order to get Ht1 candidate sequence(s), bioinformatics method was employed to analyze sequences between two flanking markers umc22a and umc122a linked with Ht1. Sixty-three open reading frame (ORF) sequences were found, fourteen of which encoded protein domains. The amino acid sequences encoded by these ORF sequences were blasted with the 24 cloned resistance gene amino acid sequences, and a phylogenetic tree was constructed. Among the fourteen ORF sequences, gpm565a with nucleotide binding site (NBS), containing a lot of random coils, had both high identity and close relationship with Xal resistant to bacterial blight, and was predicted to be related with Ht1. The remaining thirteen ORF sequences had no enough evidence to show any relationships with Ht1 due to lack of conserved domains, low identity or distant relationships.

Inducing null mutations by means of homologous recombination provides a powerful technique to investigate gene function and has found wide application in many different fields. However, it was realized some time ago that the specific way in which such knockout mutants are generated can be confounding, making it impossible to separate the effects of the induced null mutation from those of alleles originating from the embryonic stem cell donor. In addition, effects from null mutations can be altered on different genetic backgrounds. Here we present some simple breeding strategies to test for flanking gene effects that are compatible with the recommendations of the Banbury Conference on Genetic Background in Mice and with common practices of creating and maintaining mouse knockout lines.

BACKGROUND: Differential polyadenylation is a widespread mechanism in higher eukaryotes producing mRNAs with different 3' ends in different contexts. This involves several alternative polyadenylation sites in the 3' UTR, each with its specific strength. Here, we analyze the vicinity of human polyadenylation signals in search of patterns that would help discriminate strong and weak polyadenylation sites, or true sites from randomly occurring signals. RESULTS: We used human genomic sequences to retrieve the region downstream of polyadenylation signals, usually absent from cDNA or mRNA databases. Analyzing 4956 EST-validated polyadenylation sites and their -300/+300 nt flanking regions, we clearly visualized the upstream (USE) and downstream (DSE) sequence elements, both characterized by U-rich (not GU-rich) segments. The presence of a USE and a DSE is the main feature distinguishing true polyadenylation sites from randomly occurring A(A/U)UAAA hexamers. While USEs are indifferently associated with strong and weak poly(A) sites, DSEs are more conspicuous near strong poly(A) sites. We then used the region encompassing the hexamer and DSE as a training set for poly(A) site identification by the ERPIN program and achieved a prediction specificity of 69 to 85% for a sensitivity of 56%. CONCLUSION: The availability of complete genomes and large EST sequence databases now permit large-scale observation of polyadenylation sites. Both U-rich sequences flanking both sides of poly(A) signals contribute to the definition of "true" sites. However, the downstream U-rich sequences may also play an enhancing role. Based on this information, poly(A) site prediction accuracy was moderately but consistently improved compared to the best previously available algorithm.

Comparison of the sequences of mouse and human genomes revealed a surprising number of nonexonic, nonexpressed conserved sequences, for which no function could be assigned. To study the possible correlation between these conserved intronic sequences and alternative splicing regulation, we developed a method to identify exons that are alternatively spliced in both human and mouse. We compiled two exon sets: one of alternatively spliced conserved exons and another of constitutively spliced conserved exons. We found that 77% of the conserved alternatively spliced exons were flanked on both sides by long conserved intronic sequences. In comparison, only 17% of the conserved constitutively spliced exons were flanked by such conserved intronic sequences. The average length of the conserved intronic sequences was 103 bases in the upstream intron and 94 bases in the downstream intron. The average identity levels in the immediately flanking intronic sequences were 88% and 80% for the upstream and downstream introns, respectively, higher than the conservation levels of 77% that were measured in promoter regions. Our results suggest that the function of many of the intronic sequence blocks that are conserved between human and mouse is the regulation of alternative splicing.

The circadian clock exerts a major influence on transcriptional regulation in plants and other organisms. We have previously identified a motif called the evening element (EE) that is overrepresented in the promoters of evening-phased genes. Here, we demonstrate that multimerized EEs are necessary and sufficient to confer evening-phased circadian regulation. Although flanking sequences are not required for EE function, they can modulate EE activity. One flanking sequence, taken from the PSEUDORESPONSE REGULATOR 9 promoter, itself confers dawn-phased rhythms and has allowed us to define a new clock promoter motif (the morning element [ME]). Scanning mutagenesis reveals that both activators and repressors of gene expression act through the ME and EE. Although our experiments confirm that CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) are likely to act as repressors via the EE, they also show that they have an unexpected positive effect on EE-mediated gene expression as well. We have identified a clock-regulated activity in plant extracts that binds specifically to the EE and has a phase consistent with it being an activator of expression through the EE. This activity is reduced in CCA1/LHY null plants, suggesting it may itself be part of a circadian feedback loop and perhaps explaining the reduction in EE activity in these double mutant plants.

Transcriptional silencing of the gene coding for amoebapore A (AP-A) was observed when trophozoites of Entamoeba histolytica were transfected with a hybrid plasmid construct containing the ap-a gene flanked by the upstream and downstream segments of the original Ehap-a gene. Transfectants were totally devoid of ap-a transcript and AP-A protein. An identical silencing effect was observed upon transfection with a plasmid that contained only the 5' upstream region of ap-a. Removal of the selecting antibiotic enabled the isolation of plasmidless clones, which retained in their progeny the silenced phenotype. E. histolytica cells were able to overexpress ap-a when transfected with a plasmid containing the gene flanked by the 5' and 3' regions of the EhRP-L21 gene. This plasmid, however, could not express ap-a in the retransfected, cloned trophozoites lacking AP-A. This is the first report of gene silencing in E. histolytica, and the mechanism appears to belong to transcriptional gene silencing and not to posttranscriptional gene silencing. This conclusion is based on the following results:(i) silencing was achieved by transfection of homologous 5' flanking sequences (470 bp of the Ehap-a gene),(ii) transcription initiation of Ehap-a was found to be blocked, and (iii) short double-stranded RNA fragments of the ap-a coding and noncoding sequences were not detected. Trophozoites lacking AP-A are nonpathogenic and impaired in their bacteriolytic capability.

Patterns of linkage disequilibrium (LD) in the human genome are beginning to be characterized, with a paucity of haplotype diversity in "LD blocks," interspersed by apparent "hot spots" of recombination. Previously, we cloned and physically characterized the low-density lipoprotein-receptor-related protein 5 (LRP5) gene. Here, we have extensively analysed both LRP5 and its flanking three genes, spanning 269 kb, for single nucleotide polymorphisms (SNPs), and we present a comprehensive SNP map comprising 95 polymorphisms. Analysis revealed high levels of recombination across LRP5, including a hot-spot region from intron 1 to intron 7 of LRP5, where there are 109 recombinants/Mb (4882 meioses), in contrast to flanking regions of 14.6 recombinants/Mb. This region of high recombination could be delineated into three to four hot spots, one within a 601-bp interval. For LRP5, three haplotype blocks were identified, flanked by the hot spots. Each LD block comprised over 80% common haplotypes, concurring with a previous study of 14 genes that showed that common haplotypes account for at least 80% of all haplotypes. The identification of hot spots in between these LD blocks provides additional evidence that LD blocks are separated by areas of higher recombination.

Proline (Pro) is one of the most widely distributed osmolytes in water-stressed plants. We previously isolated from Arabidopsis a gene encoding Pro dehydrogenase (ProDH), a mitochondrial enzyme involved in the first step of the conversion of Pro to glutamic acid. The ProDH gene in Arabidopsis is up-regulated by rehydration after dehydration but is down-regulated by dehydration. ProDH is also induced by L-Pro and hypoosmolarity. The induction of ProDH expression under rehydration seems to be caused by both accumulated Pro and hypoosmolarity. We analyzed a DNA region that is located 5' to the transcription start site (a promoter region) of ProDH to identify cis-acting elements involved in L-Pro-induced and hypoosmolarity-induced expression in transgenic tobacco (Nicotiana tabacum) and Arabidopsis plants. We found that a 9-bp sequence, ACTCATCCT, in the ProDH promoter is necessary for the efficient expression of ProDH in response to L-Pro and hypoosmolarity. Moreover, ACTCAT is a core cis-acting element, which we have called Pro- or hypoosmolarity-responsive element (PRE), that is necessary for L-Pro-responsive and hypoosmolarity-responsive expression of ProDH. Microarray and RNA gel-blot analyses showed that 21 L-Pro-inducible genes have the PRE sequences in their promoter regions. These results indicate that the PRE sequence play an important role in the L-Pro-responsive gene expression.

AFC short interspersed elements (SINEs) were isolated from cichlids from Madagascar, the New World, and Africa and characterized. A new family of long interspersed elements (LINEs), designated the CiLINE2 family, was also isolated from African cichlids, and its consensus sequence was deduced. Upon aligning all of the consensus sequences, we found that the 3'-tail regions of the AFC SINEs and the CiLINE2 family were very similar, providing another example in which a reverse transcriptase responsible for retroposition of SINEs might be contributed in trans by a LINE. Sequence comparisons showed that CiLINE2 in cichlids was closely related to LINE2 in mammals. Furthermore, we found that the 3'-tail sequence shared by the AFC SINEs and CiLINE2 in cichlids was very similar to the 3'-tail sequence shared by the MIR SINEs and LINE2 in mammals, even though the remaining parts of the AFC SINEs and the MIR SINEs were totally different from each other. Thus, the present report not only describes a new pair of SINEs and LINEs with the same 3' tail in cichlids, but also provides a new example of the phenomenon whereby the 3' ends of LINEs with the same genealogical origin can be incorporated into the 3'-end tails of different families of SINEs that have been generated independently in two different lineages during evolution.

Gene expression is regulated by transcription factors that interact with cis-regulatory elements. Predicting these elements from sequence data has proven difficult. We describe here a successful computational search for elements that direct expression in a particular temporal-spatial pattern in the Drosophila embryo, based on a single well characterized enhancer model. The fly genome was searched to identify sequence elements containing the same combination of transcription factors as those found in the model. Experimental evaluation of the search results demonstrates that our method can correctly predict regulatory elements and highlights the importance of functional testing as a means of identifying false-positive results. We also show that the search results enable the identification of additional relevant sequence motifs whose functions can be empirically validated. This approach, combined with gene expression and phylogenetic sequence data, allows for genome-wide identification of related regulatory elements, an important step toward understanding the genetic regulatory networks involved in development.

The publication of a draft of the human genome and of large collections of transcribed sequences has made it possible to study the complex relationship between the transcriptome and the genome. In the work presented here, we have focused on mapping mRNA 3' ends onto the genome by use of the raw data generated by the expressed sequence tag (EST) sequencing projects. We find that at least half of the human genes encode multiple transcripts whose polyadenylation is driven by multiple signals. The corresponding transcript 3' ends are spread over distances in the kilobase range. This finding has profound implications for our understanding of gene expression regulation and of the diversity of human transcripts, for the design of cDNA microarray probes, and for the interpretation of gene expression profiling experiments.