Fine structure of the human ceruloplasmin gene

We characterized the genomic region corresponding to the human ceruloplasmin cDNA previously reported. Using PCR-direct sequencing methods, we determined precise intron/exon boundaries and intron-exon composition of the gene in the region. The gene region spanned about 50 kb and was composed of 19 exons and 18 introns. The lengths of exons and introns range from 107 to over 267 bp and from 0.44 to 10.0 kb, respectively. The translation initiation codon and the termination codon were located in exons 1 and 19, respectively. The nucleotide sequences of the introns were also determined in the region around the intron/exon boundaries for 24-220 bp. All the sequences around the intron/exon boundaries were consistent with the 5' and 3' consensus sequences for splice junctions of transcribed genes. Putative lariat sequences were identified between -17 and -42 nucleotides from the 3' splice junction for all 18 introns.     

Hidden Markov models for detecting remote protein homologies

MOTIVATION: A new hidden Markov model method (SAM-T98) for finding remote homologs of protein sequences is described and evaluated. The method begins with a single target sequence and iteratively builds a hidden Markov model (HMM) from the sequence and homologs found using the HMM for database search. SAM-T98 is also used to construct model libraries automatically from sequences in structural databases. METHODS: We evaluate the SAM-T98 method with four datasets. Three of the test sets are fold-recognition tests, where the correct answers are determined by structural similarity. The fourth uses a curated database. The method is compared against WU-BLASTP and against DOUBLE-BLAST, a two-step method similar to ISS, but using BLAST instead of FASTA. RESULTS: SAM-T98 had the fewest errors in all tests-dramatically so for the fold-recognition tests. At the minimum-error point on the SCOP (Structural Classification of Proteins)-domains test, SAM-T98 got 880 true positives and 68 false positives, DOUBLE-BLAST got 533 true positives with 71 false positives, and WU-BLASTP got 353 true positives with 24 false positives. The method is optimized to recognize superfamilies, and would require parameter adjustment to be used to find family or fold relationships. One key to the performance of the HMM method is a new score-normalization technique that compares the score to the score with a reversed model rather than to a uniform null model. AVAILABILITY: A World Wide Web server, as well as information on obtaining the Sequence Alignment and Modeling (SAM) software suite, can be found at http://www.cse.ucsc.edu/research/compbi o/ CONTACT: karplus@cse.ucsc.edu; http://www.cse.ucsc.edu/karplus     

The effect of UW solution and its components on the collagenase digestion of human and porcine pancreas

Miropeats displays DNA sequence similarity information graphically. The program discovers regions of similarity amongst any set of DNA sequences and then draws a graphic that summarizes the length, location and relative orientations of any repeated sequences. Sequence similarity searching is a very general tool that forms the basis of many different biological sequence analyses but it is limited by the verbosity of traditional alignment presentation styles. Miropeats enhances the utility of conventional DNA sequence comparisons when looking at long lengths of sequence similarity by summarizing large-scale sequence similarities on a single page of PostScript graphics. Miropeats has been applied estensively to help understand shotgun assembly projects, to check cosmid overlaps and to perform inter-genomic comparisons.     

GeneGenerator--a flexible algorithm for gene prediction and its application to maize sequences

MOTIVATION: We developed GeneGenerator because of the need for a tool to predict gene structure without knowing in advance how to score potential exons and introns in order to obtain the best results, pertinent in particular to less well-studied organisms for which suitable training sets are small. GeneGenerator is a very flexible algorithm which for a given genomic sequence generates a number of feasible gene structures satisfying user-defined constraints. The specific implementation described in detail requires minimum scoring for translation start and donor and acceptor splice sites according to previously trained logitlinear models. In addition, potential exons and introns are required to exceed specified minimal lengths and threshold scores for coding or non-coding potential derived as log-likelihood ratios of appropriate Markov sequence models. RESULTS: A database of 46 non-redundant genomic sequences from maize is used for illustration. It is shown that the correct gene structures do not always maximize the considered target function. However, in most cases, the correct or nearly correct structures are found in a small set of high-scoring structures. A critical review of the generated structures sometimes allows the choices to be narrowed by considering additional variables such as predicted splice site strength or local optimality of splice site scores. Summary statistics for prediction accuracy over all 46 maize genes are derived under cross-validation and non-cross-validation training conditions for the Markov sequence models. The algorithm achieved exon sensitivity of 0.81 and specificity of 0.75 on an independent set of 14 novel maize genomic segments. AVAILABILITY: GeneGenerator runs under Borland-Pascal 7.0 using MS-DOS and C on UNIX work stations. The source code is available upon request. CONTACT: jkleffe@euler.grumed.fu-berlin-de     

Genetic basis of human complement C8 alpha-gamma deficiency

Deficiency of the alpha-gamma subunit of the eighth component of complement (C8alpha-gammaD) is frequently associated with recurrent neisserial infections, especially meningitis caused by Neisseria meningitidis. We here report the molecular basis of C8alpha-gammaD in two unrelated Japanese subjects. Screening all 11 exons of the C8alpha gene and all 7 exons of the C8gamma gene and their boundaries by exon-specific PCR/single-strand conformation polymorphism demonstrated aberrant single-stranded DNA fragments in exon 2 of C8alpha gene in case 1 and in exons 2 and 9 of C8alpha gene in case 2. Nucleotide sequencing of the amplified DNA fragments in case 1 revealed a homozygous single-point mutation at the second exon-intron boundary, inactivating the universally conserved 5' splice site consensus sequence of the second intron (IVS2+1G-->T). Case 2 was a compound heterozygote for the splice junction mutation, IVS2+1G-->T, and a nonsense mutation at Arg394 (R394X). R394X was caused by a C to T transition at nucleotide 1407, the first nucleotide of the codon CGA for Arg394, leading to a stop codon TGA. No mutations were detected in the C8gamma gene by our method. Our results indicate that the pathogenesis of C8alpha-gammaD might be caused by heterogeneous molecular defects in the C8alpha gene.     

Asymmetrical recognition and activity of the I-SceI endonuclease on its site and on intron-exon junctions

Group I intron-encoded endonucleases represent a new class of double strand cutting endonucleases whose function is to initiate the homing of introns by generating double strand breaks in site-specific sequences. We have studied the mechanism of interaction of the I-SceI endonuclease with different DNA substrates derived from its natural site in the intron-less gene or from intron-exon junctions in the gene with an intron. We show that the enzyme recognizes its asymmetrical site with high affinity binding to the sequence corresponding to the downstream exon followed by binding to the upstream exon and catalysis of phosphodiester bond hydrolysis. Asymmetrical nicking activity is observed as an intermediate of the cleavage reaction. In the intron-containing gene, the enzyme recognizes the downstream intron-exon junction without any cleavage activity. This binding raises the possibility of a specific function of homing endonucleases in either gene expression or intron homing steps subsequent to DNA cleavage.     

An in vivo assay for measuring the recombination potential between DNA sequences in mammalian cells

Mammalian intermolecular recombination vectors that place the recombination junction within the intron of a selectable marker gene are presented. Many of the previously reported recombination assays require that recombination occur homologously and that they occur within the coding region of the selectable marker. This vector system involves the use of a human thymidine kinase (tk) minigene and measures the recombination frequency between any chosen DNA sequences, in mammalian thymidine kinase negative cells. The tk minigene is divided into a 5' vector and a 3' vector. In the 5' vector, the DNA sequence of interest is inserted in the proximal portion of tk intron 2. In the 3' vector, the DNA sequence of interest is inserted in the intron sequence between the proteolipid protein exon 2 and tk exons 3-7. Recombination through the DNA sequences of interest, either homologous or illegitimate, will reconstruct a functional tk minigene. The recombination junction is spliced out of the transcribed mRNA and thymidine kinase positive cells can be selected in hypoxanthine-aminopterin-thymidine medium. We have tested these vectors to measure the recombination potential of two Alu repetitive sequences (BLUR 8 and BLUR 11) against a control DNA sequence. BLUR 8 and BLUR 11 do not seem to recombine at a significantly higher frequency over that of the control DNA sequence. These recombination vectors display similar sensitivity to previous recombination systems, but allow tremendous flexibility in the choice of potentially recombinogenic sequences.     

Gene structure of the human MET proto-oncogene

By direct sequencing of cosmids using primers designed from the known cDNA sequence, we identified 19 exons in the human MET proto-oncogene, and sequenced the corresponding 5' and 3' exon-intron junctions. By homology search in the database of the Washington University Genome Sequence Center (GSC), we identified one additional exon. These 20 exons, together with a previously reported exon, bring the total exon number of MET to 21. Oligonucleotide primers were designed to amplify each exon and adjacent intronic sequences to permit examination of each exon for mutations. By restriction mapping, we assembled a 110 kb genomic contig that covered almost the entire MET proto-oncogene. This information is relevant for the screening of recently reported mutations of the MET gene which cause hereditary papillary renal carcinomas and for the search for additional mutations of the same gene which may play a role in the pathogenesis of common human carcinomas including carcinomas of the breast, ovary and pancreas.     

Resolution of Holliday junctions by RuvC resolvase: cleavage specificity and DNA distortion

E. coli RuvC protein resolves Holliday junctions during genetic recombination and postreplication repair. Using small synthetic junctions, we show that junction recognition is structure-specific and occurs in the absence of metal cofactors. In the presence of Mg2+, Holliday junctions are resolved by the introduction of symmetrically related nicks at the 3' side of thymine residues. The nicked duplex products are repaired by the action of DNA ligase. Within the RuvC-Holliday junction complex, the DNA is distorted such that 2 of the 4 strands become hypersensitive to hydroxyl radical attack. The ionic requirements of binding, hydroxyl radical sensitivity, and strand cleavage indicate three distinct steps in the mechanism of RuvC-mediated Holliday junction resolution: structure-specific recognition, DNA distortion, and sequence-dependent cleavage.     

The a sequence is dispensable for isomerization of the herpes simplex virus type 1 genome

The herpes simplex virus type 1 (HSV-1) genome consists of two components, L (long) and S (short), that invert relative to each other during productive infection to generate four equimolar isomeric forms of viral DNA. Recent studies have indicated that this genome isomerization is the result of DNA replication-mediated homologous recombination between the large inverted repeat sequences that exist in the genome, rather than site-specific recombination through the terminal repeat a sequences present at the L-S junctions. However, there has never been an unequivocal demonstration of the dispensability of the latter element for this process using a recombinant virus whose genome lacks a sequences at its L-S junctions. This is because the genetic manipulations required to generate such a viral mutant are not possible using simple marker transfer, since the cleavage and encapsidation signals of the a sequence represent essential cis-acting elements which cannot be deleted outright from the viral DNA. To circumvent this problem, a simple two-step strategy was devised by which essential cis-acting sites like the a sequence can be readily deleted from their natural loci in large viral DNA genomes. This method involved initial duplication of the element at a neutral site in the viral DNA and subsequent deletion of the element from its native site. By using this approach, the a sequence at the L-S junction was rendered dispensable for virus replication through the insertion of a second copy into the thymidine kinase (TK) gene of the viral DNA; the original copies at the L-S junctions were then successfully deleted from this virus by conventional marker transfer. The final recombinant virus, HSV-1::L-S(delta)a, was found to be capable of undergoing normal levels of genome isomerization on the basis of the presence of equimolar concentrations of restriction fragments unique to each of the four isomeric forms of the viral DNA. Interestingly, only two of these genomic isomers could be packaged into virions. This restriction was the result of inversion of the L component during isomerization, which prevented two of the four isomers from having the cleavage and encapsidation signals of the a sequence in the TK gene in a packageable orientation. This phenomenon was exploited as a means of directly measuring the kinetics of HSV-1::L-S(delta)a genome isomerization. Following infection with virions containing just the two packaged genomic isomers, all four isomers were readily detected at a stage in infection coincident with the onset of DNA replication, indicating that the loss of the a sequence at the L-S junction had no adverse effect on the frequency of isomerization events in this virus. These results therefore validate the homologous recombination model of HSV-1 genome isomerization by directly demonstrating that the a sequence at the L-S junction is dispensable for this process. The strategy used to remove the a sequence from the HSV-1 genome in this work should be broadly applicable to studies of essential cis-acting elements in other large viral DNA molecules.