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The epithelial to mesenchymal transition (EMT) involves several physiological and pathological phenomena and endows cells with invasive and migratory properties. However, the effects of substrate stiffness and topography on the migration of cells before or after transforming growth factor-β1 (TGF-β1)-induced EMT (tEMT) are unknown. Herein, we seed control or tEMT NMuMG cells on the 2D patterns consisted of 1 μm or 5 μm line-widths and groove or cone patterns on either 2 MPa (1.96 ± 0.48 MPa) or 4 MPa (3.70 ± 0.74 MPa) polydimethylsiloxane (PDMS) substrates. After tEMT, the increased expression of α-SMA with vinculin in focal adhesion (FA) sites led to an acceleration of tEMT cell motility. On the 2 MPa substrate, the most influenced substrate was the 1 μm, cone-patterned substrate, where the tEMT cells' motility decelerated by 0.13 μm/min (36% slower than the cells on groove pattern). However, on the 5 μm, groove-patterned substrate, where the tEMT cells demonstrated the most rapid motility relative to the control cells, with an increment of 0.18 μm/min (100%). Among the different physical cues from substrate, the cone pattern could impede the migration speed of tEMT cells. Furthermore, we recommend the groove-patterned with a 5 μm line-width substrate as a useful tool to differentiate control and tEMT cells by migration speed.

BackgroundGastrointestinal stromal tumors (GISTs) are specific, generally Kit (CD117)-positive, mesenchymal tumors of the gastrointestinal tract encompassing a majority of tumors previously considered gastrointestinal smooth muscle tumors. Our aim was to characterize the computed tomographic findings and predict malignant risk from computed tomography for the evaluation of GISTs.MethodsThe computed tomographic images of 39 patients with pathologically and immunohistochemically proven GISTs were reviewed by 2 radiologists, and the final interpretations were reached by consensus. Images were assessed for the size, contour, growth pattern, boundary, degree of enhancement, and necrosis of the tumors. The presence of calcification within the lesions, abdominal lymphadenopathy, ascites, and bowel obstruction were also recorded. Categorical variables were compared using Fisher's exact test. Univariate and multivariate logistic regression analyses were used for selection of significant predictors of high-risk malignancy. In addition, the relationships between computed tomographic features and tumor size were assessed by means of nonparametric univariate analysis with the Mann–Whitney U test and Kruskal–Wallis test.ResultsBoth old age and larger tumor size (≥ 5 cm) were statistically significant in the univariate logistic analysis for high-risk malignant tumors (p < 0.25). However, in multivariate logistic regression, only larger tumor size (≥ 5 cm) was found to have final statistical significance for high-risk malignant GISTs (p < 0.05). In addition, more exophytic growth pattern (p < 0.01), more lobulated appearance (p < 0.01), good enhancement (p < 0.05), and more necrosis (p < 0.01) of masses were more often observed in larger GISTs than small ones on computed tomography.ConclusionLarger tumor size (≥ 5 cm) was found to have a predictive value with respect to high-risk malignant GISTs.

A 40-year-old Taiwanese man presented with a 4-month history of intermittent fever and progressive dyspnoea on exertion. Four years previously, he had undergone surgical repair of a ruptured aneurysm of the sinus of Valsalva at Taichung Veterans General Hospital. The patient visited a local clinic with his current symptoms and was admitted for observation. Two-dimensional echocardiography revealed aortic regurgitation (AR) and mitral regurgitation (MR) in association with a prolapsed vegetation; in addition, blood culture was positive for Streptococcus viridans . He was referred to Taichung Veterans General Hospital with the diagnosis of infective endocarditis associated with AR and MR. Double valve replacement was performed and antibiotics …

We describe a novel sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 microm diameter microbeads. After constructing a microbead library of DNA templates by in vitro cloning, we assembled a planar array of a million template-containing microbeads in a flow cell at a density greater than 3x10(6) microbeads/cm2. Sequences of the free ends of the cloned templates on each microbead were then simultaneously analyzed using a fluorescence-based signature sequencing method that does not require DNA fragment separation. Signature sequences of 16-20 bases were obtained by repeated cycles of enzymatic cleavage with a type IIs restriction endonuclease, adaptor ligation, and sequence interrogation by encoded hybridization probes. The approach was validated by sequencing over 269,000 signatures from two cDNA libraries constructed from a fully sequenced strain of Saccharomyces cerevisiae, and by measuring gene expression levels in the human cell line THP-1. The approach provides an unprecedented depth of analysis permitting application of powerful statistical techniques for discovery of functional relationships among genes, whether known or unknown beforehand, or whether expressed at high or very low levels.

A common mechanism for chromosomal fragile site genesis is not yet apparent. Folate-sensitive fragile sites are expanded p(CCG)n repeats that arise from longer normal alleles. Distamycin A or bromodeoxyuridine-inducible fragile site FRA16B is an expanded AT-rich approximately 33 bp repeat; however, the relationship between normal and fragile site alleles is not known. Here, we report that bromodeoxyuridine-inducible, distamycin A-insensitive fragile site FRA10B is composed of expanded approximately 42 bp repeats. Differences in repeat motif length or composition between different FRA10B families indicate multiple independent expansion events. Some FRA10B alleles comprise a mixture of different expanded repeat motifs. FRA10B fragile site and long normal alleles share flanking polymorphisms. Somatic and intergenerational FRA10B repeat instability analogous to that found in expanded trinucleotide repeats supports dynamic mutation as a common mechanism for repeat expansion.

We describe a family in which two siblings exhibited developmental delay, reduced muscle tone and mild muscle weakness. Cytogenetic evaluation demonstrated that both children had a tandem duplication of a small portion of the long arm of chromosome 10 [46,XX or XY, dir dup(10)(q24.2{r_arrow}q24.3)], inherited from their clinically normal mother, who was found to be mosaic for the duplicated chromosome 10. Fluorescence in situ hybridization approaches, including total chromosome painting and the use of regional specific cosmid probes, were used to confirm the chromosome 10q origin of the duplicated material. This is the smallest confirmed duplication of this portion of chromosome 10 reported to date. 28 refs., 4 figs.

Uterine leiomyomata are the most common tumors in women and can cause abnormal uterine bleeding, pelvic pain, and infertility. Approximately 200,000 hysterectomies are performed annually in the U.S. to relieve patients of the medical sequelae of these benign neoplasms. Our efforts have focused on cloning the t(12;14)(q14-q15;q23-q24) breakpoint in uterine leiomyoma to further our understanding of the biology of these tumors. Thirty-nine YACs and six cosmids mapping to 12q14-q15 have been mapped by fluorescence in situ hybridization to tumor metaphase chromosomes containing a t(12;14). One YAC spanned the translocation breakpoint and was mapped to tumor metaphases from a pulmonary chondroid hamartoma containing a t(12;14)(q14-q15;q23-q24) and a lipoma containing a t(12;15)(q15;q24); this YAC also spanned the breakpoint in these two tumors, suggesting that the same gene on chromosome 12 may be involved in the pathobiology of these distinct benign neoplasms.

The human dopamine transporter gene, DAT1, acts to transport released dopamine into presynaptic terminals of the brain. The possibility that the DAT1 gene plays a role in genetic diseases of the brain has led to studies of DAT1 in several psychiatric and neurological disorders. Previous sequence analysis of DAT1 revealed a 40-bp repeat in the 3′ end of the gene. In order to identify all potential alleles for this VNTR marker a population database was established. One thousand seventy-four unrelated individuals were screened by PCR for the region containing the 40 bp repeat. Allele frequency differences were found between black Americans and Caucasians or Hispanics but no differences were observed between Caucasians and Hispanics. A previously unreported allele was detected in all three populations. Thus, we have shown that screening a large population identifies new alleles and generates more accurate allele frequencies. ©1995 Wiley-Liss, Inc.

One hundred eighty-three YACs carrying human chromosome 10 sequences were isolated from multigenome equivalent libraries by PCR-based screening for the presence of 80 different chromosome 10-specific STSs. Ninety-four of the isolated YACs, representing 52 genes and DNA segments, were mapped to regions of chromosome 10 by fluorescence in situ hybridization. The results localized 26 DNA segments to cytogenetic bands for the first time. About 37% (35/94) of the YACs hybridized to more than one chromosomal location: 31 to other chromosomes in addition to chromosome 10 and 4 to 2 distinct locations on chromosome 10. These results are consistent with the number of chimeric YACs expected from these libraries but may also reflect the presence of 2 or more YACs within a single clone or the presence of low copy repeated elements within the genome. This STS anchor screening effort resulted in the identification of 69 contigs, with 7 contigs consisting of 2 anchors each and 1 contig consisting of 5 anchors. All linked STSs were multiply linked by at least 2 independent YACs. These anchored YACs span the entire chromosome and appear to cover 15% of chromosome 10.

A total of 124 new chromosome 10-specific sequenced-tagged sites (STSs) were derived from two sources: (1) DNA sequences obtained from anonymous clones in new libraries enriched for human chromosome 10 inserts, and (2) published sequences of genes and other loci already known to map to chromosome 10. Libraries were constructed from a somatic cell hybrid carrying human chromosomes 10 and Y. A cosmid library was made from total DNA of the hybrid and probed with labeled total human DNA to identify clones with human DNA inserts. Two hundred seventeen cosmids were mapped to regions of human chromosome 10 by fluorescence in situ hybridization. Twenty-five cosmids represent probes that have been placed on the genetic map previously. One hundred ninety-two cosmids represent new probes that have not been mapped previously. Cosmids carrying inserts with CA repeats were identified by hybridization with a labeled poly-(dC-dA)-poly(dG-dT) probe and subcloned to yield microsatellite STS markers. Two small insert plasmid libraries were made, the first by subcloning inserts from a chromosome 10-enriched {lambda} phage library (LL10NS01) and the second by cloning Alu elements-mediated PCR products amplified from hybrid DNA. STSs were generated from the DNA sequences of clone inserts. Chromosome 10-specific STSs were distinguished frommore » Y chromosome STSs by one or both of the following criteria: (1) successful PCR amplification from a template consisting of DNA from another chromosome 10-containing cell line, NA10926B, or (2) FISH localization to chromosome 10 of the source cosmid or YACs isolated by PCR screening with the STS. These libraries were the source of 90 new chromosome 10-specific STSs, 42 of which contain CA repeats. 46 refs., 1 fig., 4 tabs.« less

In this study, we fabricated a photo-crosslinking, biocompatible 3D Poly(ethylene) glycol (PEG) hydrogel scaffold with inner fibrin gel for cell adhesion. We tested the material cytocompatibility with 3T3 fibroblasts then processed the cell adhesion motif inside the scaffold at regular and L-shaped 3D structural pattern by two-photon laser scanning system for further cell manipulation and recording. The results show cell could survive under UV irradiation and grew in the inner fibrin gel scaffold. The desired pattern scaffold has potential to apply in cell guided migration and physiological signal recording.

An overlapping set of 21 yeast artificial chromosomes (YACs) spanning the RET proto-oncogene [Takahashi et al., Oncogene 3 (1988) 571–578] and D10S102 markers on human chromosome 10 was isolated in a series of hybridization-based chromosomal walks in a YAC library. Genetic linkage analyses implicate this chromosomal region as the location of the gene ( MEN2A ) responsible for multiple endocrine neoplasia type 2A. Four YACs carrying a RET sequence-tagged site (STS) and two YACs carrying a D10S102 STS were used to initiate chromosome walks. These were based on hybridization of Alu element-mediated polymerase chain reaction ( Alu-PCR ) products from YACs to dot blots of Alu -PCR products from complex pools of YAC clones. The hybridization anchor content of YACs identified in the walks was confirmed by probing blots of Alu-PCR products from individual YACs and by comparing Alu-PCR fingerprints of each YAC. Ten hybridization-based Alu-PCR anchors and three STS anchors were ordered within eleven intervals created by the 21 overlapping YACs. The order of anchors requiring the fewest gaps in the YACs is consistent with the walking results and establishes the STS anchor order as D10S102-D10S94-RET . The overlapping set of YACs represents about 1.55 Mb of the human genome according to restriction mapping of four representative YACs in the contig. These results demonstrate the power of Alu-PCR hybridization for chromosomal walking and provide a rich source of overlappping YACs which can be used to identify candidate MEN2A genes.

DiGeorge (DGS, MIM 188400) and velocardiofacial (VCFS, MIM 192430) syndromes may present many clinical problems including cardiac defects, hypoparathyroidism, T-cell immunodeficiency and facial dysmorphism1. They are frequently associated with deletions within 22q11.2, but a number of cases have no detectable molecular defect of this region2,3. A number of single case reports with deletions of 10p suggest genetic heterogeneity of DGS. Here we compare the regions of hemi-zygosity in four patients with terminal deletions of 10p (one patient diagnosed as having hypoparathyroidism and three as DGS) and one patient with a large interstitial deletion (diagnosed as VCFS). Fluorescence in situ hybridization (FISH) analysis demonstrates that these patients have overlapping deletions at the 10p13/10p14 boundary. A YAC contig spanning the shortest region of deletion overlap (SRO) has been assembled, and allows the size of SRO to be approximated to 2 Mb. As with deletions of 22q11, phenotypes vary considerably between affected patients. These results strongly support the hypothesis that haploinsufficiency of a gene or genes within 10p (the DGSII locus) can cause the DGS/VCFS spectrum of malformation.

The development of noninvasive approaches to facilitate the regeneration of post-traumatic nerve injury is important for clinical rehabilitation. In this study, we investigated the effective dose of noninvasive 808-nm low-level laser therapy (LLLT) on sciatic nerve crush rat injury model. Thirty-six male Sprague Dawley rats were divided into 6 experimental groups: a normal group with or without 808-nm LLLT at 8 J/cm(2) and a sciatic nerve crush injury group with or without 808-nm LLLT at 3, 8 or 15 J/cm(2). Rats were given consecutive transcutaneous LLLT at the crush site and sacrificed 20 days after the crush injury. Functional assessments of nerve regeneration were analyzed using the sciatic functional index (SFI) and hindlimb range of motion (ROM). Nerve regeneration was investigated by measuring the myelin sheath thickness of the sciatic nerve using transmission electron microscopy (TEM) and by analyzing the expression of growth-associated protein 43 (GAP43) in sciatic nerve using western blot and immunofluorescence staining. We found that sciatic-injured rats that were irradiated with LLLT at both 3 and 8 J/cm(2) had significantly improved SFI but that a significant improvement of ROM was only found in rats with LLLT at 8 J/cm(2). Furthermore, the myelin sheath thickness and GAP43 expression levels were significantly enhanced in sciatic nerve-crushed rats receiving 808-nm LLLT at 3 and 8 J/cm(2). Taken together, these results suggest that 808-nm LLLT at a low energy density (3 J/cm(2) and 8 J/cm(2)) is capable of enhancing sciatic nerve regeneration following a crush injury.

We describe a method for cloning nucleic acid molecules onto the surfaces of 5-μm microbeads rather than in biological hosts. A unique tag sequence is attached to each molecule, and the tagged library is amplified. Unique tagging of the molecules is achieved by sampling a small fraction (1%) of a very large repertoire of tag sequences. The resulting library is hybridized to microbeads that each carry ≈10 6 strands complementary to one of the tags. About 10 5 copies of each molecule are collected on each microbead. Because such clones are segregated on microbeads, they can be operated on simultaneously and then assayed separately. To demonstrate the utility of this approach, we show how to label and extract microbeads bearing clones differentially expressed between two libraries by using a fluorescence-activated cell sorter (FACS). Because no prior information about the cloned molecules is required, this process is obviously useful where sequence databases are incomplete or nonexistent. More importantly, the process also permits the isolation of clones that are expressed only in given tissues or that are differentially expressed between normal and diseased states. Such clones then may be spotted on much more cost-effective, tissue- or disease-directed, low-density planar microarrays.

Infantile onset spinocerebellar ataxia (IOSCA) is a progressive neurological disorder of unknown etiology. It is inherited as an autosomal recessive trait and has so far been reported in just 19 Finnish patients in 13 separate families. We have previously assigned the IOSCA locus (HGMW-approved symbol SCA8) to chromosome 10q, where no previously identified ataxia loci are located. Haplotype analysis combined with genealogical data provided evidence that all the IOSCA cases in Finland originate from a single 30- to 40-generation-old founder mutation. By analyzing extended disease haplotypes observed today, the IOSCA locus can now be restricted to a region between two adjacent microsatellites, D10S192 and D10S1265, with no genetic intermarker distance. We have constructed a detailed physical map of this 270-kb IOSCA region and cytogenetically localized it to 10q24. We have also assigned two previously known genes, PAX2 and CYP17, more precisely into this region, but the sequence analysis of coding regions of these two genes has not revealed mutations in an IOSCA patient. The obtained long-range clones will form the basis for the isolation of a novel ataxia gene.

Hermansky–Pudlak syndrome (HPS) is an often–fatal autosomal recessive disease in which albinism, bleeding, and lysosomal storage result from defects of diverse cytoplasmic organelles: melanosomes, platelet dense bodies, and lysosomes. HPS is the most common single–gene disorder in Puerto Rico, with an incidence of 1 in 1,800. We have identified the HPS gene by positional cloning, and found homozygous frameshifts in this gene in Puerto Rican, Swiss, Irish and Japanese HPS patients. The HPS polypeptide is a novel transmembrane protein that is likely to be a component of multiple cytoplasmic organelles and that is apparently crucial for their normal development and function. The different clinical phenotypes associated with the different HPS frameshifts we observed suggests that differentially truncated HPS polypeptides may have somewhat different consequences for subcellular function.

Jackson-Weiss syndrome is an autosomal dominant condition characterized by craniosynostosis, foot anomalies and great phenotypic variability. Recently mutations in fibroblast growth factor receptor 2 (FGFR2) have been found in patients with another craniosynostotic syndrome, Crouzon syndrome. FGFR2 is a member of the tyrosine kinase receptor superfamily, having a high affinity for peptides that signal the transduction pathways for mitogenesis, cellular differentiation and embryogenesis. We now report an FGFR2 mutation in the conserved region of the immunoglobulin IIIc domain in the Jackson-Weiss syndrome family in which the syndrome was originally described. In addition, in four of 12 Crouzon syndrome cases, we identified two new mutations and found two previously described mutations in the same region.

Multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC) are two closely related cancer syndromes inherited in an autosomal dominant manner. Mutations in the RET proto-oncogene were found in MEN 2A and FMTC families. In this study we report seven different germline mutations in the RET proto-oncogene in five of five MEN 2A and five of six FMTC families. Each of the mutations involves a cysteine residue in the extracellular cysteine-rich domain of the RET receptor tyrosine kinase. We developed simple polymerase chain reaction based diagnostic tests for all seven mutations in these families.

Fanconi anemia is a rare autosomal recessive disorder in which affected individuals are predisposed to acute myelogenous leukemia and other malignancies. We report the results of a genetic linkage study involving 34 families enrolled in the International Fanconi Anemia Registry. A significant lod score was obtained between D20S20, an anonymous DNA segment from chromosome 20q, and Fanconi anemia ( Z max = 3.04, θ max = 0.12). However, six other anonymous DNA segments from chromosome 20q, including D20S19, which is highly polymorphic and tightly linked to D20S20, showed no or only weak evidence for linkage to Fanconi anemia. An admixture test revealed significant evidence for linkage heterogeneity ( χ 2 = 6.10, P = 0.01) at the D20S19 locus. Lod scores suggestive of linkage between Fanconi anemia and this locus were obtained with two of the largest kindreds studied (lods = 2.6 and 2.1, at θ = 0.001). Thus, our data support the provisional assignment of a Fanconi anemia gene to chromosome 20q.

A full-length cDNA copy of the mRNA encoding calf chymosin (also known as rennin), a proteolytic enzyme with commercial importance in the manufacture of cheese, has been cloned in an f1 bacteriophage vector. The nucleotide sequence of the cDNA was determined, and translation of that sequence into amino acids predicts that the zymogen prochymosin is actually synthesized in vivo as preprochymosin with a 16 amino acid signal peptide. In vitro translation of total poly(A)-enriched RNA from the calf fourth stomach (abomasum) and immunoprecipitation with antichymosin antiserum revealed that a form of chymosin (probably preprochymosin judging from the Mr-value) is the major in vitro translation product of RNA from that tissue. Gel-transfer hybridization of restriction endonuclease-cleaved bovine chromosomal DNA with labeled cDNA probes indicated that the two known forms of chymosin, A and B, must be products of two different alleles of a single chymosin gene.

We report the sequences of six S. pombe tRNA genes including two genes for tRNAArg, and one gene each for tRNAGlu, tRNAHis, tRNALys and tRNAPhe. All tRNA genes are found independently in the genome and represent individual transcription units. The gene for tRNALys has an 8 bp long intervening sequence which cannot base-pair with the tRNA anticodon. In vitro transcription studies indicate that all genes are faithfully transcribed in a yeast extract. Sequence comparison of the 5' flanking regions of the tRNA genes did not show significant homologies; however, they are very rich in AT base pairs.

Cloned tRNA genes from Drosophila and from yeast have been transcribed faithfully in extracts prepared from Xenopus germinal vesicles. The newly formed RNA is composed of precursor tRNAs (of 5S RNA size) and of tRNAs. The plasmid pCIT12 carries genes for Drosophila tRNALys, tRNAArg, and tRNAAsn, Nucleotide analysis of one RNA species transcribed from pCIT12 DNA showed it to be identical to Drosophila tRNALys; it even contained some of the modified nucleotides expected for this tRNA. This RNA species is formed in the germinal vesicle extract via a larger precursor tRNA molecule that does not contain nucleotide modifications. This simple transcription system should aid studies aimed at defining the regulatory DNA regions responsible for eukaryotic gene transcription. In addition, it may provide tRNA precursors that are needed for detailed investigations of eukaryotic tRNA biosynthesis.

The genes for two new P-type ATPases, PMR1 and PMR2, have been identified in yeast. A comparison of the deduced sequences of the PMR proteins with other known ion pumps showed that both proteins are very similar to Ca2+ ATPases. PMR1 is identical to SSC1, a gene previously identified by its effect on secretion of some foreign proteins from yeast. Proteins secreted from pmr1 mutants lack the outer chain glycosylation that normally results from passage through the Golgi. Loss of PMR1 function suppresses the lethality of ypt1-1, a mutation that blocks the secretion pathway. These data suggest that PMR1 functions as a Ca2+ pump affecting transit through the secretory pathway.

Using oligonucleotide probes with defined sequences, we have selected clones from a human lymphocyte cDNA library which represent human leukocyte (HuIFN-alpha) and fibroblast (HuIFN-beta) interferon gene sequences. Double-stranded f1 phage DNA was used as the vector for initial cloning of cDNA. Clones carrying interferon gene sequences were identified by hybridization with the oligonucleotide probes. The same oligonucleotide probes were used as primers for dideoxy chain termination sequencing of the clones. One HuIFN-alpha clone, 201, has a nucleotide sequence different from published HuIFN-alpha sequences. Under control of the lacUV5 promoter, the 201 gene has been used to express biologically active HuIFN-alpha in Escherichia coli.

Sequence analysis of a Schizosaccharomyces pombe DNA fragment revealed two tRNA coding regions separated by a seven nucleotide spacer. the 5'-proximal tRNA gene encodes a tRNAUCGSer sequence, which is interrupted by a 16 nucleotide intron at the 3' side of the base adjacent to the anticodon. The second tRNA gene encodes an initiator tRNAMet sequence. This DNA fragment, cloned into pBR322, was used as template for in vitro transcription in a nuclear extract of Xenopus oocytes. The tRNA genes were transcribed into one RNA precursor which contained both tRNA sequences. The primary transcription product initiates with pppG, contains a 9 nucleotide leader sequence, a 16 nucleotide intron, a 7 nucleotide spacer between the two tRNA molecules and an 8--9 nucleotide trailer sequence. RNA initiation was only observed upstream of the 5'-proximal tRNASer. We used RNA analysis to establish a sequence of the enzymatic steps of tRNA maturation in the nuclear extract. The first step in processing the dimeric precursor is an endonuclease cleavage which generates the mature 5' end of the tRNAMet. Further steps include the removal of the flanking sequences and addition of the CCAOH 3' terminus. The last step is the splicing of the tRNASer precursor to remove the intervening sequence.

Extracts from Xenopus germinal vesicles have been used to transcribe faithfully cloned Drosophila or yeast tRNA genes to precursor tRNAs. If the tRNA genes contain an intervening DNA sequence it is found in the tRNA precursors as the corresponding RNA sequence. The Xenopus nuclear extracts have the ability to process these precursor tRNAs into mature tRNA. Sequence analysis of the primary transcripts, which contain 5′-terminal pppA or pppG residues, should aid studies aimed at defining regulatory DNA regions responsible for gene transcription by RNA polymerase III. In addition, these tRNA precursors have proven to be ideal substrates for the many enzymes involved in eukaryotic tRNA biosynthesis.

Two DNA fragments, each containing tRNA(Arg)3 and a tRNA(Asp) gene in close conjunction, have been isolated from different genomic regions of Saccharomyces cerevisiae. Nucleotide Nucleotide sequence analysis of the gene regions revealed that in both fragments the tRNA(Arg)3 coding region is located 5'-proximal to the tRNA(Asp) coding region. They are separated by an identical spacer of 10 nucleotides. Although the 5'-flanking sequences are different in the two plasmids, some similarities are observed. To test the mode of expression of this gene configuration, we transcribed the DNA fragments in a Xenopus oocyte nuclear extract. Specific transcription of the yeast tRNA genes took place in an RNA precursor which comprised both tRNA species. We report here that the precursor RNA was processed to the mature-sized tRNA molecules, indicating the presence of an enzyme activity in the Xenopus nucleus capable of cutting a dimeric tRNA precursor. This is the first observation of a eukaryotic dimeric tRNA precursor.

Three Schizosaccharomyces pombe dimeric tRNA genes, consisting of a tRNASer gene encoding a minor species with an intervening sequence followed by a tRNAMeti gene, have been described [Mao et al. (1980) Cell 21, 509-516; Hottinger et al. (1982) Mol. Gen. Genet. 188, 219-224; Willis et al. (1984) EMBO J. 3, 1573-1580]. We have examined the reason for the dimeric structure by comparing the transcriptional efficiencies and competitive abilities of the genes subcloned from the dimeric arrangement. Both of the subcloned genes are active in vivo in Saccharomyces cerevisiae, but only the tRNASer gene is efficiently transcribed in vitro. The tRNASer gene competes efficiently for transcription factors, while the tRNAMeti gene does so only weakly. Thus, it appears that the dimeric arrangement is required to support expression of the tRNAMeti gene. S. pombe genes encoding major species of tRNASer are transcribed considerably less efficiently than are the minor genes from the dimers, so coupling of the tRNAMeti gene to the minor species genes should lead to efficient production of tRNAMeti.

We have characterized the rRNA gene repeat in Schizosaccharomyces pombe. This repeat, which does not contain the 5S RNA gene, is found in a 10.4 kb HindIII DNA fragment. We have determined the nucleotide sequences of the S. pombe 5.8S RNA gene and intergenic spacers from two different 10.4 kb DNA fragments. Analysis of isolated total cellular 5.8S RNA revealed the presence of eight species of 5.8S RNA, differing in the number of nucleotides at the 5'-end. The eight 4.8S RNA species vary in length from 158 to 165 nucleotides. Apart from the heterogeneity observed at the 5'-end, the sequence of the eight 5.8S RNA species appears to be identical and is the same sequence as coded for by the 5.8S genes. The gene sequence shows great homology to the 5.8S RNA genes or S. cerevisiae and N. crassa. Most of the base differences are confined to the highly variable stem though to be involved in co-axial helix stacking with the 25S RNA, where base pairing is nearly identical despite the sequence differences. Secondary structure models are examined in light of 5.8S RNA oligonucleotide conservation across species from yeasts to higher eukaryotes.

The genomic arrangement and sequences of S. pombe 5S RNA genes are reported here. The 5S gene sequences appear to be dispersed within the genome, and are found independently of other rRNA genes. The sequences of two 5S genes examined show identical coding regions of 119 base pairs but have widely varying flanking sequences. A tRNAAsp gene is found in the 3' flanking region of one of the 5S genes. The tRNAAsp gene is faithfully transcribed in an X. laevis in vitro system, while the 5S genes are not transcribed in this system. The phylogenetic position of S. pombe is examined through comparison of 5S RNA sequences.

The nucleotide sequence of 1.5 Mb of genomic DNA fromMycobacterium leprae was determined using computer-assisted multiplex sequencing technology. This brings the 2.8-Mb M. leprae genome sequence to ∼66% completion. The sequences, derived from 43 recombinant cosmids, contain 1046 putative protein-coding genes, 44 repetitive regions, 3 rRNAs, and 15 tRNAs. The gene density of one per 1.4 kb is slightly lower than that ofMycoplasma (1.2 kb). Of the protein coding genes, 44% have significant matches to genes with well-defined functions. Comparison of 1157 M. leprae and 1564 Mycobacterium tuberculosisproteins shows a complex mosaic of homologous genomic blocks with up to 22 adjacent proteins in conserved map order. Matches to known enzymatic, antigenic, membrane, cell wall, cell division, multidrug resistance, and virulence proteins suggest therapeutic and vaccine targets. Unusual features of the M. leprae genome include large polyketide synthase (pks) operons, inteins, and highly fragmented pseudogenes.[The sequence data described in this paper have been submitted to GenBank under accession nos. L78811–L78829,U00010–U00023, U15180–U15184, U15186, U15187, L01095, L01536, L04666, and L01263. On-line supplementary information for Table 1 is available at http://www.cshl.org/gr.]