Td Gene Research Articles

Whole-exome sequencing identifies high-confidence genes for tic disorders in a Chinese Han population

BackgroundTic disorder (TD) is a polygenic neurodevelopmental disorder with high susceptibility. However, identifying high-confidence risk genes has been challenging due to poor replication across multiple studies. MethodsWhole-exome sequencing was performed on 390 TD patients and 372 unaffected individuals in a Chinese Han population. Analysis of variance, burden analysis and in silico prediction were used to identify candidate genes for TD. To facilitate data analysis and to focus on high-confidence genes, we defined a panel of 160 genes as known causal or candidate TD genes from previous studies. Gene enrichment and protein–protein interaction analysis were utilized to detect potential novel TD risk genes. ResultsTotally, 14 variants across 12 known TD candidate genes were considered potential susceptibility variants. Ten variants across 10 known TD candidate genes were identified as potential disease-causing variants. Burden analysis identified variants of 28 known genes were significantly excess in TD patients. In addition, 354 previously unproven TD genes are over-represented in patients. Genes enriched in the PI3K-Akt signaling, sphingolipid metabolism and serotonergic synaptic pathways, as well as those interacting with FN1, were considered potential new candidate genes for TD. ConclusionsThis is the largest WES study focusing on TD patients in a Chinese Han population. Several variants recurring in our cohort were identified as high-confidence risk loci for TD. Moreover, we provided potential new risk genes that may be prioritized for further investigation.

An Improved Model for Circular RNA Overexpression: Using the Actin Intron Reveals High Circularization Efficiency.

Traditionally, the group 1 intron of the T4 td gene is used to generate a foreign circular sequence. However, the T4 system has been shown to be fairly inefficient in expressing circular RNA (circRNA). Here, a new method is developed to express circular sequences with high circularization efficiency to strengthen the confidence for future circRNA functional studies. CircRNA expression plasmids, constructed with different lengths derived from the actin intron (15-nt, 30-nt, 60-nt, 100-nt, 180-nt) and T4 intron, are introduced into human and mouse cell lines 293T and B16. Junction detection and sequencing are used to determine successful circularization of introns and their expression efficiencies. An actin intron with a medium length (60-nt-100-nt) shows significantly increased efficiency of circularization, whereas intron-100-nt shows the best efficiency in most conditions. RNA pull-down assays are designed to precipitate the splicing factors that are bound to the introns and intron/exon junction. The precipitated proteins are analyzed by mass spectrometry (MS), aiming to identify the possible underlying mechanism behind the high circularization efficiency. This expression system has been validated using different circRNAs, and such method shows potential in contributing to the expanding field of circRNA studies.

Loss-of-Function Mutation of Soybean R2R3 MYB Transcription Factor Dilutes Tawny Pubescence Color.

Pubescence color of soybean is controlled by two genes, T and Td. In the presence of a dominant T allele, dominant and recessive alleles of the Td locus generate tawny and light tawny (or near-gray) pubescence, respectively. Flavones, responsible for pubescence color, are synthesized via two copies of flavone synthase II genes (FNS II-1 and FNS II-2). This study was conducted to map and clone the Td gene. Genetic and linkage analysis using an F2 population and F3 families derived from a cross between a Clark near-isogenic line with light tawny pubescence (genotype: TT tdtd) and a Harosoy near-isogenic line with tawny pubescence (TT TdTd) revealed a single gene for pubescence color around the end of chromosome 3. Genome sequence alignment of plant introductions revealed an association between premature stop codons in Glyma.03G258700 (R2R3 MYB transcription factor) and recessive td allele. Cultivars and lines having near-gray or light tawny pubescence and a gray pubescence cultivar with td allele had premature stop codons in the gene. These results suggest that Glyma.03G258700 corresponds to the Td gene. It was predominantly expressed in pubescence. Compared to a tawny pubescence line, a near-isogenic line with td allele produced extremely small amounts of transcripts of Glyma.03G258700, FNS II-1, and FNS II-2 in pubescence. The promoter of FNS II-1 and FNS II-2 shared cis-acting regulatory elements for binding of MYB proteins. These results suggest that the wild type of Glyma.03G258700 protein may bind to the promoter of FNS II genes and upregulate their expression, resulting in increased flavone content and deeper pubescence color. In contrast, mutated Glyma.03G258700 protein may fail to upregulate the expression of FNS II genes, resulting in decreased flavone content and dilute pubescence color.

Abstract 5411: Curcumin nanoformulation: A new therapeutic approach for cervical cancer treatment

Abstract Background: Cervical cancer is one of the most common and deadly cancers among women worldwide and is associated with persistent Human Papillomavirus (HPV) infection. Currently, Cisplatin based chemo and radio therapy is the best standard treatment for cervical cancer. While screening for cervical cancer can reduce the incidence and mortality, treatment of advanced stage cervical cancer is difficult and often unsuccessful. Resistance to chemo-radio therapy with prolonged treatment, resulting in an invasive form of cancer, requires the development of novel therapeutic modalities to conquer chemo resistance and improve overall life expectancy of patients. Nanotechnology provides targeted delivery of anti-cancer drugs and indicates a new approach for cancer diagnosis and treatment. Our lab has developed PLGA (poly [lactic-co-glycolic acid]), an FDA approved polymer coated nanoparticle(s) using a well-known anti-cancer drug Curcumin (CUR) for the enhanced bioavailability and improved therapeutic efficacy of CUR. Therefore, “we hypothesized that having targeted drug delivery, curcumin nanoparticles (NC) will show improved anti-cancerous effects on cervical cancer cells and will also overcome drug (Cisplatin) resistance when compared with free curcumin.” Methods: The effect of NC on the proliferation of Caski cervical cancer cells was determined by MTS and colony formation assay. Annexin V/7AAD and propidium iodide (PI) staining was analyzed by flow cytometry to determine NC's effect on apoptosis and cell cycle inhibition, respectively. The expression of apoptotic markers such as poly (ADP-ribose) polymerase (PARP), Caspase 3 and Caspase 9 was determined by immunoblotting. The potential of NC to modulate the expression of miRNAs associated with drug resistance was determined by quantitative RT-PCR. In order to assess efficacy of NC to reduce tumor growth, an orthotopic mouse model was generated using Caski cells stably transfected with fluorescent gene td tomato. Results: NC effectively inhibited Caski cell growth and also induced apoptosis as determined by staining for Annexin V/7AAD and immunoblotting for cleaved PARP, Caspase 3, and Caspase 9. PI staining clearly revealed that NC treatment arrests growth cycle of Caski cells in the G1- S phase. Additionally, NC treatment caused a marked decrease in the levels of miRNA 21, an oncomiRNA associated with chemoresistance, and enhanced miRNA 214, a tumor suppressor, when compared to free Curcumin. Moreover, NC formulation effectively reduces the tumor burden in athymic nude mice. Conclusion: Our findings show that PLGA based NC significantly inhibits Caski cervical cancer cell growth and regulates the expression of miRNAs, associated with cervical cancer. In vivo experiments show that NC is efficacious in reducing the tumor burden. Therefore, NC may be a novel chemo-preventive and therapeutic modality for the overall management of cervical cancer. Citation Format: Mohd Saif Zaman, Neeraj Chauhan, Diane M. Maher, Murali M. Yallapu, Meena Jaggi, Subhash C. Chauhan. Curcumin nanoformulation: A new therapeutic approach for cervical cancer treatment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5411. doi:10.1158/1538-7445.AM2014-5411

Changes in Free Amino Acid Content in ‘Jonagold’ Apple Fruit as Related to Branched-chain Ester Production, Ripening, and Senescence

The relationship among the free amino acid content, the expression of genes related to branched-chain amino acid metabolism {branched-chain aminotransferase [BCAT], α-keto acid decarboxylase [pyruvate decarboxylase (PDC)], and threonine deaminase [TD]}, and the production of branched-chain (BC) esters during ripening and senescence in ‘Jonagold’ apple fruit (Malus ×domestica) was studied. Eighteen amino acids were measured by liquid chromatography coupled with tandem mass spectrometry. The content for all amino acids changed with developmental stage and some shared similar patterns of accumulation/diminution. The pattern for isoleucine differed from all other amino acids, increasing more than 20-fold during the ripening process. The onset of the increase was concomitant with the onset of increasing ethylene and BC ester production and the content remained elevated even during senescence. The elevated isoleucine levels are consistent with an increase in the flux through the pathway leading to the formation and degradation of the isoleucine precursor α-keto-β-methylvalerate, which is used for production of BC esters containing 2-methylbutanol and 2-methylbutanoate moieties. Unexpectedly, the content of threonine, the amino acid from which isoleucine is thought to be derived in plants, did not change in concert with isoleucine, but rather declined somewhat after ripening was well underway. Patterns in the expression of some, but not all, of the putative BCAT and PDC genes appeared to reflect the rise and fall in ester formation; however, the expression of putative TD genes did not change during ripening. The patterns in gene expression and amino acid content are interpreted to suggest that the synthesis of α-keto-β-methylvalerate and isoleucine during apple ripening may depend on an as yet uncharacterized pathway that bypasses threonine, similar to the citramalate pathway found in some bacteria.

Gene expression analyses of hepatocellular adenoma and hepatocellular carcinoma from the marine flatfish Limanda limanda

At selected sites around the UK, the offshore sentinel flatfish species dab Limanda limanda are found to contain elevated levels of macroscopic liver tumors. Previous proteomic and metabolomic studies have demonstrated that differences exist between tumor and non-tumor tissues; however, these differing features were not identified, and little is known about the changes at the gene expression level, or whether prognostic markers are present and can be identified. A flounder Platichthys flesus custom cDNA microarray and RT-PCR were used to investigate hepatic mRNA expression in the histologically confirmed tumors, hepatocellular adenoma (HA) and hepatocellular carcinoma (HC) from dab, and in adjacent normal tissue from the same fish. Differences in gene expression were observed between tumor and normal tissues, and between tumor types. A class-prediction approach using 50 transcripts revealed sufficient group-specific expression profiles to allow segregation of samples dependent on their tumor type or the sex of the host. Vitellogenins were found to display the greatest induction (up to 500-fold induction) in some HC tumors from female fish and in both HA and HC tumors from males. To the best of our knowledge, this is the first report of the association of vitellogenin expression with tumors of wild fish.

Scientific Serendipity Initiates an Intron Odyssey

As the child of German immigrants to South Africa and disillusioned by the Apartheid regime, I made my way to the United States for graduate school. After earning my Ph.D. from the University of California at Irvine and a postdoctoral degree at the Hebrew University in Jerusalem, both in phage λ genetics, I faced a five-body problem: two careers and three children. Two positions and a family-friendly environment brought us to the Capital District in New York. For me, it was a research scientist position, working on thymidylate synthase (TS). Even in the 1970s, TS was a well studied enzyme, highly regulated in each organism in which it had been studied, and above all, TS was a geneticist's dream: positive selections for both gene function and dysfunction in bacteria. So, I set to work with respected biochemists Frank and Gladys Maley, and almost 30 years ago, we published our first paper together on a single functional arginine involved in TS catalysis (1). This arginine was to reappear on my research radar in a completely different context more than 20 years later. Meanwhile, I developed the TS genetic system for studying the enzyme's function and regulation (2) and taught my colleague Fred Chu how to sequence the TS coding sequence (the td gene) of phage T4. Sequencing was a big deal in those days; entire papers were dedicated to decoding a single gene, and I had taken a week-long course to learn how to sequence DNA. Chu did a superb job, but he presented us with a conundrum. The td gene contained a chunk of sequence that did not correspond to TS. Without the benefit of GenBank™ and BLAST searches, we were left to ponder for ourselves what that intervening sequence might be: an artifact? An intron? But dogma insisted that introns existed only in eukaryotic genes.

Analysis of Flavonoids in Flower Petals of Soybean Near-isogenic Lines for Flower and Pubescence Color Genes

W1, W3, W4, and Wm genes control flower color, whereas T and Td genes control pubescence color in soybean. W1, W3, Wm, and T are presumed to encode flavonoid 3'5'-hydroxylase (EC 1.14.13.88), dihydroflavonol 4-reductase (EC 1.1.1.219), flavonol synthase (EC 1.14.11.23), and flavonoid 3'-hydroxylase (EC 1.14.13.21), respectively. The objective of this study was to determine the structure of the primary anthocyanin, flavonol, and dihydroflavonol in flower petals. Primary component of anthocyanin in purple flower cultivars Clark (W1W1 w3w3 W4W4 WmWm TT TdTd) and Harosoy (W1W1 w3w3 W4W4 WmWm tt TdTd) was malvidin 3,5-di-O-glucoside with delphinidin 3,5-di-O-glucoside as a minor compound. Primary flavonol and dihydroflavonol were kaempferol 3-O-gentiobioside and aromadendrin 3-O-glucoside, respectively. Quantitative analysis of near-isogenic lines (NILs) for flower or pubescence color genes, Clark-w1 (white flower), Clark-w4 (near-white flower), Clark-W3w4 (dilute purple flower), Clark-t (gray pubescence), Clark-td (near-gray pubescence), Harosoy-wm (magenta flower), and Harosoy-T (tawny pubescence) was carried out. No anthocyanins were detected in Clark-w1 and Clark-w4, whereas a trace amount was detected in Clark-W3w4. Amount of flavonols and dihydroflavonol in NILs with w1 or w4 were largely similar to the NILs with purple flower suggesting that W1 and W4 affect only anthocyanin biosynthesis. Amount of flavonol glycosides was substantially reduced and dihydroflavonol was increased in Harosoy-wm suggesting that Wm is responsible for the production of flavonol from dihydroflavonol. The recessive wm allele reduces flavonol amount and inhibits co-pigmentation between anthocyanins and flavonols resulting in less bluer (magenta) flower color. Pubescence color genes, T or Td, had no apparent effect on flavonoid biosynthesis in flower petals.

RNA chaperone activity of the Sm-like Hfq protein.

The Escherichia coli Sm-like host factor I (Hfq) protein is thought to function in post-transcriptional regulation by modulating the function of small regulatory RNAs. Hfq also interferes with ribosome binding on E. coli ompA messenger RNA, indicating that Hfq also interacts with mRNAs. In this study, we have used stimulation of group I intron splicing in vivo and a modified in vitro toeprinting assay to determine whether Hfq acts as an RNA chaperone. Hfq was able to rescue an RNA 'folding trap' in a splicing defective T4 bacteriophage td gene in vivo. Enzymatic analysis showed that Hfq affects the accessibility of the ompA start codon, as well as other bases within the ribosome-binding site, explaining its negative effect on ribosome binding. We also show that the Hfq-induced structural changes in ompA mRNA are maintained after proteolytic digestion of the protein, which classifies Hfq as an RNA chaperone.

Mouse Tdho abnormality results from double point mutations of the emopamil binding protein gene (Ebp).

Mouse Tdho (Tattered-Hokkaido) was described as being allelic with Td in our previous study. Both allelic genes, which are located at the same position on the centromere of the X Chromosome (Chr), generate similar phenotypes such as male embryonic lethality, and in heterozygous females, hyperkeratotic skin, skeletal abnormalities, and growth retardation. The emopamil binding protein gene (Ebp) emerged as a candidate for mouse Tdho mutation, since the Td gene was recently determined to result from a point mutation of Ebp. In this study, Ebp cDNA of Tdho was demonstrated to possess double point mutations that cause two amino acid changes from Leu to Pro at position 132 and from Ser to Cys at 133 in EBP protein. EBP participates in cholesterol biosynthesis, and cholest-8(9)-en-3beta-ol was found to be increased in the plasma of Tdho adult females but not in that of normal mice. From these results, a loss of function was expected for the EBP protein encoded by Tdho. Both the phenotypes and genes responsible for Tdho as well as Td are quite similar to those of human X-linked chondrodysplasia punctata (CDPX2).

A Transcript Map of a 2-Mb BAC Contig in the Proximal Portion of the Mouse X Chromosome and Regional Mapping of the scurfy Mutation

A physical clone contig has been constructed, spanning 2 Mb on the proximal mouse X chromosome containing the mouse scurfy (sf) and tattered (Td) mutations. Extensive transcript mapping in this interval has identified 37 potential transcription units, including a number of novel genes, and 4 pseudogenes. These genes have been ordered by STS content and restriction mapping. Comparison of the transcript map to the corresponding region in human Xp11.23–p11.22 shows extensive homology, with complete conservation of gene order for loci in common between the two maps. Further, using a novel method to identify simple sequence length polymorphisms, we have developed a number of genetic markers, which has enabled the region containing the sf mutation to be narrowed to <300 kb. This contig has already allowed the cloning of the Td gene using a candidate gene approach and now serves as a starting point for the cloning of the sf mutation.

Assaying RNA chaperone activity in vivo using a novel RNA folding trap.

In the absence of proteins, RNAs often misfold in vitro due to alternative base pairings which result from the molecule being trapped in inactive conformations. We identify an in vivo folding trap in the T4 phage td gene, caused by nine base pairs between a sequence element in the upstream exon of the td gene and another at the 3' end of the intron. During translation, the ribosome resolves this interaction; consequently the intron folds correctly and splicing occurs. The introduction of a stop codon upstream of this base pairing prevents resolution of the inactive structure so that splicing cannot proceed. We have used this folding trap to probe for RNA binding proteins which, when overexpressed, either resolve the misfolded structure or impede its formation in vivo. We distinguish between proteins which recognize the intron structure and those which bind non-specifically and apparently ignore the intron. The first class, e.g. Neurospora crassa CYT-18, can rescue the exonic trap and intron mutants which cause a structural defect. However, known RNA chaperones such as Escherichia coli StpA and S12 and the HIV protein NCp7, only resolve the exonic trap without suppressing intron mutations. Thus, this structural trap enables detection of RNA chaperone activity in vivo.

Recombination-dependent DNA replication stimulated by double-strand breaks in bacteriophage T4.

We analyzed the mechanism of recombination-dependent DNA replication in bacteriophage T4-infected Escherichia coli using plasmids that have sequence homology to the infecting phage chromosome. Consistent with prior studies, a pBR322 plasmid, initially resident in the infected host cell, does not replicate following infection by T4. However, the resident plasmid can be induced to replicate when an integrated copy of pBR322 vector is present in the phage chromosome. As expected for recombination-dependent DNA replication, the induced replication of pBR322 required the phage-encoded UvsY protein. Therefore, recombination-dependent plasmid replication requires homology between the plasmid and phage genomes but does not depend on the presence of any particular T4 DNA sequence on the test plasmid. We next asked whether T4 recombination-dependent DNA replication can be triggered by a double-strand break (dsb). For these experiments, we generated a novel phage strain that cleaves its own genome within the nonessential frd gene by means of the I-TevI endonuclease (encoded within the intron of the wild-type td gene). The dsb within the phage chromosome substantially increased the replication of plasmids that carry T4 inserts homologous to the region of the dsb (the plasmids are not themselves cleaved by the endonuclease). The dsb stimulated replication when the plasmid was homologous to either or both sides of the break but did not stimulate the replication of plasmids with homology to distant regions of the phage chromosome. As expected for recombination-dependent replication, plasmid replication triggered by dsbs was dependent on T4-encoded recombination proteins. These results confirm two important predictions of the model for T4-encoded recombination-dependent DNA replication proposed by Gisela Mosig (p. 120-130, in C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berget (ed.), Bacteriophage T4, 1983). In addition, replication stimulated by dsbs provides a site-specific version of the process, which should be very useful for mechanistic studies.

Bacteriophage T4 gene 28

The complete nucleotide sequence of bacteriophage T4D gene 28 has been determined. Gene 28 product is a structural component of the viral baseplate for which an enzymatic activity has also been proposed.

Synthesis of circular RNA in bacteria and yeast using RNA cyclase ribozymes derived from a group I intron of phage T4.

Studies on the function of circular RNA and RNA topology in vivo have been limited by the difficulty in expressing circular RNA of desired sequence. To overcome this, the group I intron from the phage T4 td gene was split in a peripheral loop (L6a) and rearranged so that the 3' half intron and 3' splice site are upstream and a 5' splice site and 5' half intron are downstream of a single exon. The group I splicing reactions excise the internal exon RNA as a circle (RNA cyclase ribozyme activity). We show that foreign sequences can be placed in the exon and made circular in vitro. Expression of such constructs (RNA cyclase ribozymes) in Escherichia coli and yeast results in the accumulation of circular RNA in these organisms. In yeast, RNA cyclase ribozymes can be expressed from a regulated promoter like an mRNA, containing 5' leader and 3' trailer regions, and a nuclear pre-mRNA intron. RNA cyclase ribozymes have broad application to questions of RNA structure and function including end requirements for RNA transport or function, RNA topology, efficacy of antisense or ribozyme gene control elements, and the biosynthesis of extremely long polypeptides.

Distribution and characterization of mutations induced by nitrous acid or hydroxylamine in the intron-containing thymidylate synthase gene of bacteriophage T4

The detailed distribution and characterization of 51 hydroxylamine (HA)-induced and 59 nitrous acid (NA)-induced mutations in the intron-containing bacteriophage T4 thymidylate synthase (td) gene is reported here. Mutations were mapped in 10 regions of the td gene by recombinational marker rescue using plasmid or M13 subclones of the td gene. Phage crosses using deletion mutants with known breakpoints in the 3' end of the td intron subdivided HA and NA mutations which mapped in this region. At least 31 of the mutations map within the 1-kb group I self-splicing intron. Intron mutations mapped only in the 5' and 3' ends of the intron sequence, in accordance with the hypothesis that the 5' and 3' domains of the T4 td intron are essential for correct RNA splicing. RNA sequence analysis of a number of mapped td mutations has identified two intron nucleotides and one exon nucleotide where both HA- and NA-induced mutations commonly occur. These three loci are characterized by a GC dinucleotide, with the mutations occurring at the cytosine residue. Thus, these data indicate at least three potential sites of both HA- and NA-induced mutagenic hotspot activity within the td gene.

A proflavin-induced frameshift hotspot in the thymidylate synthase gene of bacteriophage T4

Twenty-one independent thymidylate synthase deficient ( td) mutants were isolated after proflavin mutagenesis of T4D o phage. A strikingly high proportion of these mutations (17 of 21; 80%) mapped in a small 122 nucleotide (nt) region which spans the 5′ splice site of this intron-containing gene. This region comprises only 14% of the total td exon sequence. RNA sequence analysis of these mutants identified a series of frameshift insertion/deletion mutations and indicated a hotspot for proflavin-induced mutations in the 3′ end of exon I of the td gene. The mutant sequences at the hotspot site fully support a previously proposed mutagenic mechanism for proflavin-induced mutations in which frameshifts are produced as a consequence of exonuclease or DNA polymerase activity at the 3′ ends of nicks in the DNA produced by perturbation of the T4-encoded type II topoisomerase activity by the acridine. Sixteen of the seventeen DNA mutations in the hotspot region can be explained by the model as a consequence of enzymatic processing of nicks at two phospodiester bonds staggered by 4 base pairs (bp) and located on opposite strands of the DNA. Thus, these mutants exhibit precisely the symmetry expected of topoisomerase-mediated mutagenesis. The DNA sequences of the td hotspot mutants, when considered with the sequences of proflavin-induced mutants in the T4 rIIB and lysozyme genes, confirm the view that proflavin-induced mutations in diverse bacteriophage T4 DNA sequences are all produced by the topoisomerase-dependent mechanisms and do not support the view that classical misalignments in DNA repeats are hotspots for proflavin-induced mutagenesis in T4.

Biosynthetic threonine deaminase gene of tomato: isolation, structure, and upregulation in floral organs.

The gene encoding the plant biosynthetic threonine deaminase (Td; EC 4.2.1.16) has been cloned as a result of its unusual upregulation in tomato flowers. The Td gene of tomato encodes a polypeptide of 595 residues, the first 80 of which comprise a putative two-domain transit peptide cleaved at position 51. Comparison of the amino acid sequence with the corresponding enzymes from yeast and bacteria reveals a near identity of the important catalytic regions and greater than 40% overall similarity. The Td gene is unique in the tomato genome and its coding region is interrupted by eight introns. Its expression is greater than 50-fold higher in sepals and greater than 500-fold higher in the rest of the flower than in leaves or roots. Its overexpression, however, is strictly confined to the parenchymal cells of the floral organs. In young tomato leaves, the chloroplast-bound enzyme is found almost exclusively in the subepidermal spongy mesophyll cells.

Purification and substrate specificity of a T4 phage intron-encoded endonuclease

The T4 phage td intron-encoded endonuclease (I-Tev I) cleaves the intron-deleted td gene (td delta I) 23 nucleotides upstream of the intron insertion site on the noncoding strand and 25 nucleotides upstream of this site on the coding strand, to generate a 2-base hydroxyl overhang in the 3' end of each DNA strand. I-Tev I-157, a truncated form in which slightly more than one third (88 residues) of the endonuclease is deleted, was purified to homogeneity and shown to possess endonuclease activity similar to that of I-TEV I, the full-length enzyme (245 residues). The minimal length of the td delta I gene that was cleaved by I-Tev I and I-Tev I-157 has been determined to be exactly 39 basepairs, from -27 (upstream in exon1) to +12 (downstream in exon2) relative to the intron insertion site. Similar to the full-length endonuclease, I-Tev I-157 cuts the intronless thymidylate synthase genes from such diverse organisms as Escherichia coli, Lactobacillus casei and the human. The position and nature of the in vitro endonucleolytic cut in these genes are homologous to those in td delta I. Point mutational analysis of the td delta I substrate based on the deduced consensus nucleotide sequence has revealed a very low degree of specificity on either side of the cleavage site, for both the full-length and truncated I-TEV I.

Spontaneous shuffling of domains between introns of phage T4

The three self-splicing introns in phage T4 (in the td, sunY and nrdB genes) (Fig. 1a) each have the conserved group I catalytic RNA core structure (Fig. 1b), out of which is looped an open reading frame. Although the core sequences are very similar (approximately 60% identity), the open reading frames seem to be unrelated. Single crossover recombination events between homologous core sequences in the closely linked td and nrdB introns have led to 'exon shuffling. Here we describe spontaneous double crossovers between the unlinked td and sun Y introns that result in shuffling of an intron structure element, P7.1 (refs 3 and 4). The intron domain-switch variants were isolated as genetic suppressors of a splicing-defective P7.1 deletion in the td intron. This unprecedented example of suppression through inter-intron sequence substitution indicates that the introns are in a state of genetic flux and implies the functional interchangeability of the two analogous but nonidentical P7.1 elements. The implications of such recombination events are discussed in the light of the evolution of the introns themselves as well as that of their host genomes.