Human Intron Research Articles

Generalized tetracycline induced Cre recombinase expression through the ROSA26 locus of recombinant mice

Inducible Cre recombinase systems have been developed to bypass initial lethal phenotypes and to provide access to later embryonic or adult phenotypes. Here we describe the generation of a recombinant mouse that combines a tetracycline dependent switch with generalized Cre recombinase expression by targeting the ubiquitously expressed ROSA26 locus. This transgenic strain was developed using a simplified gene delivery system integrating both elements, the reverse tetracycline controlled trans-activator (rtTA) and rtTA inducible promoter into a single vector. In this transgenic strain, the endogenous ROSA26 promoter drives rtTA expression through a splice acceptor site. The tetracycline inducible promoter, cloned in opposite orientation to the ROSA26 locus and separated from the rtTA element by a 5kb human p53 intron, drives Cre recombinase expression. Crossing these mice with a Cre reporter strain showed that Cre DNA-mediated recombination was ubiquitously and effectively induced during various prenatal developmental windows. Background Cre recombinase expression levels were observed in some tissues in the absence of the inducer, mostly during late embryonic developmental stages and in adult animals. Background recombination levels were low during development and most prominent in nervous tissue. Cre recombinase expression could not be effectively induced in adult animals. While rtTA mRNA levels were high in developmental and adult tissues, Cre recombinase mRNA levels remained low after doxycycline treatment. The mouse strain described here provides a valuable tool to further analyze the function of genes during specific developmental windows, by allowing the effective inactivation of their function throughout defined stages of embryonic development.

Human and mouse introns are linked to the same processes and functions through each genome's most frequent non-conserved motifs

We identified the most frequent, variable-length DNA sequence motifs in the human and mouse genomes and sub-selected those with multiple recurrences in the intergenic and intronic regions and at least one additional exonic instance in the corresponding genome. We discovered that these motifs have virtually no overlap with intronic sequences that are conserved between human and mouse, and thus are genome-specific. Moreover, we found that these motifs span a substantial fraction of previously uncharacterized human and mouse intronic space. Surprisingly, we found that these genome-specific motifs are over-represented in the introns of genes belonging to the same biological processes and molecular functions in both the human and mouse genomes even though the underlying sequences are not conserved between the two genomes. In fact, the processes and functions that are linked to these genome-specific sequence-motifs are distinct from the processes and functions which are associated with intronic regions that are conserved between human and mouse. The findings show that intronic regions from different genomes are linked to the same processes and functions in the absence of underlying sequence conservation. We highlight the ramifications of this observation with a concrete example that involves the microsatellite instability gene MLH1.

Splicing Factor SPF30 Bridges an Interaction between the Prespliceosome Protein U2AF35 and Tri-small Nuclear Ribonucleoprotein Protein hPrp3

Spliceosome assembly is a dynamic process involving the sequential recruitment and rearrangement of small nuclear ribonucleoproteins (snRNPs) on a pre-mRNA substrate. Here we identify several spliceosome protein interactions with different domains of human splicing factor SPF30 that have the potential to mediate the addition of the tri-snRNP to the prespliceosome. In particular, we show that the C-terminal tails of SmD1, SmD3, and the protein Lsm4 interact with the central Tudor domain of SPF30. We identify a novel interaction between the N-terminal domain of SPF30 and U2AF35, a prespliceosome protein that has a role in recognizing the 3' splice site and recruiting U2 snRNP. We also show that the C terminus of SPF30 interacts with a middle domain of hPrp3, a component of U4/U6 di-snRNP and the tri-snRNP. Importantly, we show that the U2AF35 and hPrp3 interactions with SPF30 can occur simultaneously, thereby potentially linking 3' splice site recognition with tri-snRNP addition. Finally, we note that SPF30 and its partner-interacting domains are not conserved in yeast, suggesting this interaction network may play an important role in the complex splicing observed in higher eukaryotes.

Extremely Intron-Rich Genes in the Alveolate Ancestors Inferred with a Flexible Maximum-Likelihood Approach

Chromalveolates are a large, diverse supergroup of unicellular eukaryotes that includes Apicomplexa, dinoflagellates, ciliates (three lineages that form the alveolate branch), heterokonts, haptophytes, and cryptomonads (three lineages comprising the chromist branch). All sequenced genomes of chromalveolates have relatively low intron density in protein-coding genes, and few intron positions are shared between chromalveolate lineages. In contrast, genes of different chromalveolates share many intron positions with orthologous genes from other eukaryotic supergroups, in particular, the intron-rich orthologs from animals and plants. Reconstruction of the history of intron gain and loss during the evolution of chromalveolates using a general and flexible maximum-likelihood approach indicates that genes of the ancestors of chromalveolates and, particularly, alveolates had unexpectedly high intron densities. It is estimated that the chromalveolate ancestor had, approximately, two-third of the human intron density, whereas the intron density in the genes of the alveolate ancestor is estimated to be slightly greater than the human intron density. Accordingly, it is inferred that the evolution of chromalveolates was dominated by intron loss. The conclusion that ancestral chromalveolate forms had high intron densities is unexpected because all extant unicellular eukaryotes have relatively few introns and are thought to be unable to maintain numerous introns due to intense purifying selection in their, typically, large populations. It is suggested that, at early stages of evolution, chromalveolates went through major population bottlenecks that were accompanied by intron invasion.

Identification of motifs that function in the splicing of non-canonical introns

The enrichment of specific intronic splicing enhancers upstream of weak PY tracts suggests a novel mechanism for intron recognition that compensates for a weakened canonical pre-mRNA splicing motif.

Alternative Modes of Binding by U2AF65 at the Polypyrimidine Tract

During initial recognition of an intron in pre-mRNA, the 3' end of the intron is bound by essential splicing factors. Notably, the consensus RNA sequences bound by these proteins are highly degenerate in humans. This raises the question of 3' splicing factor function in introns lacking canonical binding sites. Investigating the introns of the model organism Neurospora crassa revealed a different organization at the 3' end of the intron compared to most eukaryotic organisms. The predicted branch point sequences of Neurospora introns are much closer to the 3' splice site compared to those in human introns. In addition, Neurospora introns lack the canonical polypyrimidine tract found at the end of introns in most eukaryotic organisms. The large subunit of the U2 snRNP associated factor (U2AF65), which is essential for splicing of human introns and specifically recognizes the polypyrimidine tract, is also present in Neurospora. We show that Neurospora U2AF65 binds RNA with low affinity and specificity, apparently evolving with its disappearing binding site. The arginine/serine rich domain at the N-terminus of Neurospora U2AF65 regulates its RNA binding. We find that this regulated binding can be recapitulated in human U2AF65 which has been mutated to decrease both affinity and overall charge. Finally, we show that the addition of the small U2AF subunit (U2AF35) to U2AF65 with weakened RNA binding affinity significantly enhances the affinity of the resulting U2AF heterodimer.

Transposition of two amino acids changes a promiscuous RNA binding protein into a sequence-specific RNA binding protein

In yeast (Saccharomyces cerevisiae), the branchpoint binding protein (BBP) recognizes the conserved yeast branchpoint sequence (UACUAAC) with a high level of specificity and affinity, while the human branchpoint binding protein (SF1) binds the less-conserved consensus branchpoint sequence (CURAY) in human introns with a lower level of specificity and affinity. To determine which amino acids in BBP provide the additional specificity and affinity absent in SF1, a panel of chimeric SF1 proteins was tested in RNA binding assays with wild-type and mutant RNA substrates. This approach revealed that the QUA2 domain of BBP is responsible for the enhanced RNA binding affinity and specificity displayed by BBP compared with SF1. Within the QUA2 domain, a transposition of adjacent arginine and lysine residues is primarily responsible for the switch in RNA binding between BBP and SF1. Alignment of multiple branchpoint binding proteins and the related STAR/GSG proteins suggests that the identity of these two amino acids and the RNA target sequences of all of these proteins are correlated.

Novel Single Nucleotide Polymorphisms in the Promoter and Intron 1 of Human Pregnane X Receptor/NR1I2 and Their Association with CYP3A4 Expression

The hypothesis was tested that sequence diversity in pregnane X receptor (PXR) cis-regulatory regions is a significant determinant of variation in inducible and constitutive CYP3A4 expression. A combination of comparative genomics and computational algorithms was used to select regions of the human PXR promoter and intron 1 that were resequenced in the polymorphism discovery resource 24 DNA subset. PXR single nucleotide polymorphisms (SNP) were then genotyped in donor human livers phenotyped for CYP3A4 and multidrug resistance protein 1 mRNA and primary human hepatocytes phenotyped for basal and rifampin-inducible CYP3A4 activity. The human PXR promoter [16.9 kilobase (kb)] was significantly larger than in rodents (2.9 kb). Eighty-nine SNPs were identified in the promoter and intron 1 of PXR. The SNPs most consistently associated with CYP3A4 phenotypic measures were a 44477T>C(-1359) promoter SNP (in linkage disequilibrium with SNP 463970, a 6-base pair deletion in intron 1a, and SNP 46551, a C nucleotide insertion in intron 1b); SNP 63396C>T in intron 1 (in linkage disequilibrium with SNP 63704A>G, a 63813(CAAA)(CA) variable repeat, and SNP 65104T>C); and SNP 56348C>A, SNP 69789A>G, and SNP 66034T>C. Donor livers with the variant PXR alleles had altered hepatic expression of PXR targets compared with livers with PXR wild-type alleles. These results identified PXR promoter and intron 1 SNPs associated with PXR target gene expression (CYP3A4) in donor livers and cultured hepatocytes and that a striking number of the linked intron 1 SNPs will affect putative binding sites for hepatic nuclear factor 3beta (FOXA2), a transcription factor linked with PXR expression.

Molecular Study of (TG)m(T)n Polymorphisms in Iranian Males With Congenital Bilateral Absence of the Vas Deferens

Congenital bilateral absence of the vas deferens (CBAVD) is a frequent cause of obstructive azoospermia. Nearly 75% of men with CBAVD have at least 1 detectable common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The different alleles at the (TG)(m)(T)(n) polymorphic locus at the 3' end of human CFTR intron 8 determine the efficiency of exon 9 splicing. To study the CFTR gene mutations and (TG)(m)(T)(n) polymorphisms in Iranian CBAVD patients with presumed low CF frequency and to better understand the complex regulation of exon 9 splicing among our study population, we analyzed CFTR mutations and (TG)(m)(T)(n) polymorphisms in 112 Iranian CBAVD, 7 congenital unilateral absence of the vas deferens males from Iran, and 84 fertile males as controls. Moreover, we compared the rate of CFTR transcripts with exon 9 (9+) with reduction of the (T)(n) repeat in our study population. Our study showed that the 5T mutation was present with high frequency in our patients. Longer (TG)(m) polymorphic tracts increase the proportion of exon 9 deletion transcripts but only when activated by the 5T allele. The combination of the 5T allele in 1 copy of the CFTR gene with a CF mutation in the other copy is the most common cause of CBAVD in the Iranian population. We also observed the highest level of exon 9+ splicing efficiency among the tested samples with the (TG)(12)(T)(7) allele, which represents the most common intron 8 splice variant allele in the general population. Our results support the idea that a putative role of the (T)(n) repeat is to distance the (TG)(m) repeat from the 3' splice site and that the different alleles at the (T)(n) locus affect the efficiency by which the splice acceptor consensus sequence is recognized.

Erythroid-Specific Expression of β-Globin by the Sleeping Beauty Transposon for Sickle Cell Disease

Sickle cell disease (SCD) results predominately from a single monogenic mutation that affects thousands of individuals worldwide. Gene therapy approaches have focused on using viral vectors to transfer wild-type beta- or gamma-globin transgenes into hematopoietic stem cells for long-term expression of the recombinant globins. In this study, we investigated the use of a novel nonviral vector system, the Sleeping Beauty (SB) transposon (Tn) to insert a wild-type beta-globin expression cassette into the human genome for sustained expression of beta-globin. We initially constructed a beta-globin expression vector composed of the hybrid cytomegalovirus (CMV) enhancer chicken beta-actin promoter (CAGGS) and full-length beta-globin cDNA, as well as truncated forms lacking either the 3' or 3' and 5' untranslated regions (UTRs), to optimize expression of beta-globin. Beta-globin with its 5' UTR was efficiently expressed from its cDNA in K-562 cells induced with hemin. However, expression was constitutive and not erythroid-specific. We then constructed cis SB-Tn-beta-globin plasmids using a minimal beta-globin gene driven by hybrid promoter IHK (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human alphaLCR, ankyrin-1 promoter), IHbetap (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human alphaLCR, beta-globin promoter), or HS3betap (HS3 core element from human betaLCR, beta-globin promoter) to establish erythroid-specific expression of beta-globin. Stable genomic insertion of the minimal gene and expression of the beta-globin transgene for >5 months at a level comparable to that of the endogenous gamma-globin gene were achieved using a SB-Tn beta-globin cis construct. Interestingly, erythroid-specific expression of beta-globin driven by IHK was regulated primarily at the translational level, in contrast to post-transcriptional regulation in non-erythroid cells. The SB-Tn system is a promising nonviral vector for efficient genomic insertion conferring stable, persistent erythroid-specific expression of beta-globin.

Pitfalls of PCR-Based Strategy for Genotyping Cre-Loxp Mice

Pitfalls of PCR-Based Strategy for Genotyping <i>Cre-Loxp</i> Mice

Alternative splicing and bioinformatic analysis of human U12-type introns

U12-type introns exist, albeit rarely, in a variety of multicellular organisms. Splicing of U12 intron-containing precursor mRNAs takes place in the U12-type spliceosome that is distinct from the major U2-type spliceosome. Due to incompatibility of these two spliceosomes, alternative splicing involving a U12-type intron may give rise to a relatively complicated impact on gene expression. We studied alternative U12-type intron splicing in an attempt to gain more mechanistic insights. First, we characterized mutually exclusive exon selection of the human JNK2 gene, which involves an unusual intron possessing the U12-type 5′ splice site and the U2-type 3′ splice site. We demonstrated that the long and evolutionary conserved polypyrimidine tract of this hybrid intron provides important signals for inclusion of its downstream alternative exon. In addition, we examined the effects of single nucleotide polymorphisms in the human WDFY1 U12-type intron on pre-mRNA splicing. These results provide mechanistic implications on splice-site selection of U12-type intron splicing. We finally discuss the potential effects of splicing of a U12-type intron with genetic defects or within a set of genes encoding RNA processing factors on global gene expression.

Effect of variants in the ovine skeletal-muscle-specific calpain gene on body weight

The ovine skeletal-muscle-specific calpain gene (p94), which is known also as the n-calpain or calpain 3 gene (CAPN3), was screened with primers. Selection of the PCR primers was based on the ovine cDNA sequence (GenBank accession No. AF087570). After sequence alignment between the ovine and human (AY902237) genes, exon and intron boundaries were determined. Polymorphisms were observed in the intron region for the CAPN31112 and CAPN31213 segments, and the sequences for these segments were submitted to the GenBank (AF309635 and AY102617, respectively). Body weight was recorded at birth, weaning and post-weaning. Calpain 3 genotypes of the CAPN31112 segment were associated with birth weight (P < 0.01), and a dominant gene effect was observed. Breeding group, birth type, and rearing type were significantly associated with weight traits. Allele frequencies were similar in purebred and crossbred animals.

Modern origin of numerous alternatively spliced human introns from tandem arrays

Despite the widespread occurrence of spliceosomal introns in the genomes of higher eukaryotes, their origin remains controversial. One model proposes that the duplication of small genomic portions could have provided the boundaries for new introns. If this mechanism has occurred recently, the 5' and 3' boundaries of each resulting intron should display distinctive sequence similarity. Here, we report that the human genome contains an excess of introns with perfect matching sequences at boundaries. One-third of these introns interrupt the protein-coding sequences of known genes. Introns with the best-matching boundaries are invariably found in tandem arrays of direct repeats. Sequence analysis of the arrays indicates that many intron-breeding repeats have disseminated in several genes at different times during human evolution. A comparison with orthologous regions in mouse and chimpanzee suggests a young age for the human introns with the most-similar boundaries. Finally, we show that these human introns are alternatively spliced with exceptionally high frequency. Our study indicates that genomic duplication has been an important mode of intron gain in mammals. The alternative splicing of transcripts containing these intron-breeding repeats may provide the plasticity required for the rapid evolution of new human proteins.

Most recent AluY insertions in human gene introns reduce the content of the primary transcripts in a cell type specific manner

Being the most effectively transposed primate-specific SINEs, Alu elements are present in more than one million copies in the human genome and include most recently transposed subsets of AluY elements that are polymorphic in humans. Although Alu elements are commonly thought to play an essential role in shaping and functioning of primate genomes, the understanding of the impact of recent Alu insertions on human gene expression is far from being comprehensive. Here we compared hnRNA contents for allele pairs of genes heterozygous for AluY insertions in their introns in human cell lines of various origins. We demonstrated that some AluY insertions correlated with decreased content of the corresponding hnRNAs. The effect observed does not depend on sequences of Alu elements and their orientation but is likely to be cell type specific.

Long L1 insertions in human gene introns specifically reduce the content of corresponding primary transcripts

LINE-1 (L1) retrotransposons comprise about 17% of the human genome and include a recently transposed set of Ta-L1 elements that are polymorphic in humans. Although it is widely believed that L1s play an essential role in shaping and functioning of mammalian genomes, the understanding of the impact of L1 insertions on gene expression is far from being comprehensive. Here we compared hnRNA contents for allele pairs of genes heterozygous for Ta-L1 insertions in their introns in human cell lines of various origin. We demonstrated that some Ta-L1 insertions correlated with decreased content of the corresponding hnRNAs. This effect was characteristic of only nearly full-sized L1s and seemed to be tissue specific.

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BioTechniquesVol. 41, No. 2 WebWatchOpen AccessWebWatchKevin AhernKevin AhernSearch for more papers by this authorPublished Online:21 May 2018https://doi.org/10.2144/000112227AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Inside InformationThe general scheme of signal transduction is pretty simple—information outside the cell is conveyed inside to elicit a cellular response. Many extracellular signaling molecules interact with cell surface receptors and initiate an internal signal, without ever entering the cell. Others, like steroid hormones, however, are invaders that dock with protein shepherds in the cytoplasm. These ligand-bound receptors move to the nucleus where they initiate transcription of genes important for many cellular processes. The Nuclear Receptor dataBASE (NuReBase) provides access to data on this fascinating group of receptor proteins. Search options for retrieving gene sequences or gene families (estrogen receptors, for example) help to make NuReBase a valuable tool for researchers investigating the process of signaling.@ www.ens-lyon.fr/LBMC/laudet/nurebase/nurebase.htmlEatabugadayHumans have a love/hate relationship with insects. Universally, we adore butterflies, but aside from entomologists, no one likes ants at a picnic, house flies, or mosquitoes buzzing after dark on a warm summer night. We design chemicals to poison the pests and use UV lights and pheromones to entice them to fly into high voltage screens. We're not alone in having mixed feelings about chitinous creatures. Many plants depend on winged species for pollination, yet others see insects as dinner. The Carnivorous Plants web site tracks the “meat eaters” with an information-loaded set of pages. If you thought the Venus Fly Trap was the only carnivorous plant, you're in for a surprise. From the Pitcher plants of the southeast, to the Darlingtonia of the Pacific Northwest, carnivorous plants are surprising beauties to behold and friends to insect haters everywhere.@ www.honda-e.comBiocopiaMaybe you want a job in the biotech industry. Perhaps you are trying to troubleshoot a pesky experimental protocol in molecular biology. You might be searching for databases, educational content, or literature specific to microarrays, but no matter what aspect of biological science interests you, there's a good chance you'll find it at Biowww.net. Packed tightly into Biowww's hierarchically organized opening page are links to subject sites that span topics ranging from neurosciences and immunology to jobs and journals, as well as a methods database. There are resources for mass spectrometrists, tools for drug discovery, protocols for site-directed mutagenesis, and even euthanasia guidelines for lab animals. It doesn't matter if your bag is molecular biology, pharmacology, protein chemistry, immunology, electron microscopy, or cell biology, there's something at Biowww almost guaranteed to tickle your fancy.@ biowww.netOut of AfricaOriginally developed as a part of Dr. Tulio de Oliveira's Ph.D. thesis at the University of Pretoria, BioAfrica has a mission—”to organize and distribute information on the HIV/AIDS subtype C epidemic in Africa.” Numerous re sources support this effort, including epi demiological information, country-by-country fact sheets, a phylogenetic tree of 116 sequences, software for BLASTing, and miscellaneous pieces of information relating to the serotype's prevalence and infection rate. Visitors can download software products, perform proteomics analyses, and get miscellaneous information on workshops, maps, and reports. Like a one-stop shop, BioAfrica has something for everyone.@ www.bioafrica.netChoice CutsOne of the surprises of the human genome was the relatively small number of genes discovered. We needn't fear a protein diversity shortage, however, as the slicing and dicing of mRNAs via alternative splicing gives rise to hundreds of thousands of different combinations. Keeping on top of the multiple forms of the 30,000+ delicately carved human (and other species’) transcripts is the job of the Alternative Splicing Database (ASD) of the European Bioinformatics Institute, where visitors can examine alternate splice junctions in the AltSplice annotated database or sequences of alter native human exons and introns in the AltExtron database. From sequences to functional analyses to disease information, ASD has it covered.@ www.ebi.ac.uk/asd/index.htmlFiguresReferencesRelatedDetails Vol. 41, No. 2 Follow us on social media for the latest updates Metrics History Published online 21 May 2018 Published in print August 2006 Information© 2006 Author(s)PDF download

Bioinformatic discovery of microRNA precursors from human ESTs and introns

BackgroundMicroRNAs (miRNAs) function in many physiological processes, and their discovery is beneficial for further studying their physiological functions. However, many of the miRNAs predicted from genomic sequences have not been experimentally validated to be authentic expressed RNA transcripts, thereby decreasing the reliability of miRNA discovery. To overcome this problem, we examined expressed transcripts – ESTs and intronic sequences – to identify novel miRNAs as well as their target genes.ResultsTo facilitate our approach, we developed our scanning method using criteria based on the features of 207 known human pre-miRNAs to discriminate miRNAs from random sequences. We identified 208 candidate hairpins in human ESTs and human reference gene intronic sequences, 52 of which are known pre-miRNAs. The discovery pipeline performance was further assessed using 130 newly updated pre-miRNA and randomly selected sequences. We achieved sensitivity of 85% (110/130) and overall specificity of 49.7% using this method. Because miRNAs are evolutionarily conserved regulators of gene expression, it is expected that their host genes and target genes should have respective phylogenetic orthologs. Our results confirmed that, in certain mammals, the host genes carrying the same miRNAs are orthologs, as previously reported. Moreover, this observation is also the case for some of the miRNA target genes.ConclusionWe have predicted 208 human pre-miRNA candidates and over 10,000 putative human target genes. Using sequence information from ESTs and introns ensures that the predicted pre-miRNA candidates are expressed and the combined expression transcription information from ESTs and introns makes our prediction results more decisive with regard to expressed pre-miRNAs.

On the Estimation of Intron Evolution

PLoS Computational Biology recently published an article about spliceosomal intron evolution by Nguyen, Yoshihama, and Kenmochi [1]. The authors were unaware of some earlier independent results. Foremostly, the main point of the article—that of estimating the density of potential intron sites—is not novel. It was described more than three months earlier [2]. The numerical results are virtually identical in the two publications, which is not surprising, since they apply the same model to the same data [3]. A recent article points to the model's validity. Raible and coauthors [4] report that introns in the protostome Platynereis dumerilii are almost as abundant as in humans, and many introns are in homologous positions between the two species. The shared positions indicate that at most one-third of human introns were gained in the vertebrate lineage, in agreement with the estimates of [2] and [1]. In contrast, parsimony estimates [3] should change significantly when including P. dumerilii. To estimate ancestral intron losses and gains, Nguyen and coauthors use an exponential-time procedure, which is practical only for a few species. In reality, the estimation can be done in linear time [2], as described briefly below. We are modeling intron presence and absence in homologous sites across organisms related by a known phylogeny. Presence and absence are encoded by 1 and 0, respectively. Introns evolve independently, by a Markov model for a binary character. On branch e, an intron is lost with probability pe(1 → 0) and an intron is gained with probability pe(0 → 1) at every site. Assuming a continuous-time Markov process, where λ, μ > 0 are branch-specific gain and loss rates, and t > 0 is branch length. Introns are observed at the terminal taxa. An all-absent intron site is never observed, and, thus, the number of potential intron sites must be estimated for correct likelihood optimization. The likelihood can be computed by a dynamic programming algorithm [5]. The algorithm calculates the conditional likelihood Lu(x) for every node u and state x ∈ {0,1}: Lu(x) is the probability of the observed states in descendants of u, conditioned on the state x at u. One can further define the upper conditional likelihood Uu(x) for the observed states outside the subtree of u, which can be computed efficiently by dynamic programming even if the underlying process is irreversible [2]. Feslenstein [6] reviews relevant techniques for the reconstruction of ancestral molecular sequences, which are generally assumed to evolve by a reversible process. Now, the posterior probability of the intron state x at every node u can be computed as The posterior probability for state change x → y on an edge uv is computed as The expected numbers of gains or losses are obtained by summing the probabilities quv(0 → 1) and quv(1 → 0) over all intron sites, respectively. Nguyen and coauthors consider instead all 2N state labeling of N internal nodes to compute the expected numbers of gains and losses. A Java package implements the more efficient procedure, and is publicly available at http://www.iro.umontreal.ca/~csuros/introns/. Nguyen et al. [1] reiterate well-known concerns of identifiability. Their Proposition 1 echoes the Pulley Principle for ambiguous root placement [5]. Proposition 2 asserts that there are two possible parameter sets pe(x → y) for every branch, which can be combined to get exponentially many choices that give the same likelihood function. The continuous-time process of Equation 1 implies pe(0 → 1) + pe(1 → 0) < 1. Such constraint leads to unique parametrization (except for the root position), and is more natural than the one proposed by Nguyen and coauthors, which is based on the variance of intron gains and losses. Nguyen and coauthors discuss an important study by Qiu, Schisler, and Stoltzfus [7]. Qiu and coauthors constructed multiple alignments of ten gene families. The families had 68 sequences and 49 intron sites on average. Using a Bayesian framework, Qiu and coauthors estimated two intron evolution parameters per family, assuming constant rates across sites and branches. The model's adequacy and some of the conclusions can certainly be debated, especially in view of the assumption of constant rates. Nguyen and coauthors, however, speculate that the data were insufficient for valid inference, since there are 268 possible intron presence–absence patterns for the average gene family, but only 49 intron sites. The argument is not sound: the number of patterns has little to do with inference (consider the case of a protein alignment with 20k possible patterns for k sequences). It is the number of parameters that matters.

Comparative genomic mapping of the bovine Fragile Histidine Triad (FHIT) tumour suppressor gene: characterization of a 2 Mb BAC contig covering the locus, complete annotation of the gene, analysis of cDNA and of physiological expression profiles

BackgroundThe Fragile Histidine Triad gene (FHIT) is an oncosuppressor implicated in many human cancers, including vesical tumors. FHIT is frequently hit by deletions caused by fragility at FRA3B, the most active of human common fragile sites, where FHIT lays. Vesical tumors affect also cattle, including animals grazing in the wild on bracken fern; compounds released by the fern are known to induce chromosome fragility and may trigger cancer with the interplay of latent Papilloma virus.ResultsThe bovine FHIT was characterized by assembling a contig of 78 BACs. Sequence tags were designed on human exons and introns and used directly to select bovine BACs, or compared with sequence data in the bovine genome database or in the trace archive of the bovine genome sequencing project, and adapted before use. FHIT is split in ten exons like in man, with exons 5 to 9 coding for a 149 amino acids protein. VISTA global alignments between bovine genomic contigs retrieved from the bovine genome database and the human FHIT region were performed. Conservation was extremely high over a 2 Mb region spanning the whole FHIT locus, including the size of introns. Thus, the bovine FHIT covers about 1.6 Mb compared to 1.5 Mb in man. Expression was analyzed by RT-PCR and Northern blot, and was found to be ubiquitous. Four cDNA isoforms were isolated and sequenced, that originate from an alternative usage of three variants of exon 4, revealing a size very close to the major human FHIT cDNAs.ConclusionA comparative genomic approach allowed to assemble a contig of 78 BACs and to completely annotate a 1.6 Mb region spanning the bovine FHIT gene. The findings confirmed the very high level of conservation between human and bovine genomes and the importance of comparative mapping to speed the annotation process of the recently sequenced bovine genome. The detailed knowledge of the genomic FHIT region will allow to study the role of FHIT in bovine cancerogenesis, especially of vesical papillomavirus-associated cancers of the urinary bladder, and will be the basis to define the molecular structure of the bovine homologue of FRA3B, the major common fragile site of the human genome.