Insertion of a rabbit beta-globin gene sequence into an E. coli plasmid.

Double stranded DNA has been synthesized in vitro from rabbit globin messenger RNA and elongated with homopolymeric dG tails. An E. coli plasmid was cleaved by EcoRI. The cohesive ends were repaired and dC tails added, to permit reconstitution of the EcoRI sites upon annealing with the dG elongated globin DNA. Transformation of E. coli with the globin-plasmid DNA hybrid has yielded a clone which harbours a recombinant plasmid (pCR1-betaG1), as demonstrated by hybridization experiments with radioactive globin cDNA. The sequence carried by the recombinant plasmid corresponds to part of the gene sequence coding for the beta chain of rabbit globin. Circular DNA of the purified recombinant plasmid exhibits sensitivity to EcoRI.     

A memory-efficient dynamic programming algorithm for optimal alignment of a sequence to an RNA secondary structure

Background  

Covariance models (CMs) are probabilistic models of RNA secondary structure, analogous to profile hidden Markov models of linear sequence. The dynamic programming algorithm for aligning a CM to an RNA sequence of length N is O(N ³) in memory. This is only practical for small RNAs.     

Severin, gelsolin, and villin share a homologous sequence in regions presumed to contain F-actin severing domains

cDNA clones encoding the actin filament severing protein severin from Dictyostelium discoideum were isolated from a cDNA library in lambda gt 11 using monoclonal antibodies. Comparison of the deduced amino acid sequence with the sequence of a severin peptide indicated that the complete coding region of severin is contained in the isolated clones. Severin, a 39.9-kDa protein, is encoded by one gene in D. discoideum. An mRNA of approximately 1.4 kilobases is present throughout the developmental cycle of D. discoideum. The amino acid sequence of severin contains a region highly homologous to a conserved sequence in villin and gelsolin, two proteins of similar function isolated from vertebrates. This homologous region is believed to participate in the actin filament severing activity of these proteins. Comparison of the severin sequence to the entire gelsolin sequence shows remarkable homologies pointing to a common origin from an ancestral gene from which gelsolin has been derived by a duplication.     

Inferring an organism-specific optimal threshold for predicting protein coding regions in eukaryotes based on a bootstrapping algorithm

The accuracy of prediction methods based on power spectrum analysis depends on the threshold that is used to discriminate between protein coding and non-coding sequences in the genomes of eukaryotes. Because the structure of genes vary among different eukaryotes, it is difficult to determine the best prediction threshold for a eukaryote relying only on prior biological knowledge. To improve the accuracy of prediction methods based on power spectral analysis, we developed a novel method based on a bootstrap algorithm to infer organism-specific optimal thresholds for eukaryotes. As prior information, our method requires the input of only a few annotated protein coding regions from the organism being studied. Our results show that using the calculated optimal thresholds for our test datasets, the average prediction accuracy of our method is 81%, an increase of 19% over that obtained using the same empirical threshold P = 4 for all datasets. The proposed method is simple and convenient and easily applied to infer optimal thresholds that can be used to predict coding regions in the genomes of most organisms.     

Estimating a founder's genomic proportion for each descendant in an outbred pedigree.

The question of how to estimate a founder's proportion of a single descendant's genome has renewed relevance for outbred pedigrees, given the abundant DNA sequence data for model and nonmodel eukaryotes alike. Here we show that a donor-recurrent shortcut method can provide a robust estimation of founder proportions. In addition, we define the theoretical variance and estimate confidence intervals using a nonparameteric bootstrap method. Using actual marker data from a highly heterozygous outbred Pinus taeda pedigree, it was found that each founder's genomic proportion varied widely for each descendant, ranging from 8.8% to 38.7%. In 1 case, skewed transmission of a founder's genome could be statistically detected. Its founder proportions ranged from 1.54% to 48.46%, and its mean value was 17.59%, well below the expected value of 25%. Two-thirds of its 91 descendants had 1 or the other founder haplotype, despite 2 successive meioses. The donor-recurrent method was robust; variation for estimated founder's proportions was also wide for simulated high-density datasets whether markers were dispersed or clustered. Estimating founder contributions using this computational shortcut has broad application for highly heterozygous outbred pedigrees characterized by large sibships, low population differentiation, and shallow physical mapping resources. The relevance of this computational shortcut for outbred populations used for conservation, domestication, and evolutionary biology research is discussed.     

Efficient identification of DNA hybridization partners in a sequence database

MOTIVATION: The specific hybridization of complementary DNA molecules underlies many widely used molecular biology assays, including the polymerase chain reaction and various types of microarray analysis. In order for such an assay to work well, the primer or probe must bind to its intended target, without also binding to additional sequences in the reaction mixture. For any given probe or primer, potential non-specific binding partners can be identified using state-of-the-art models of DNA binding stability. Unfortunately, these models rely on dynamic programming algorithms that are too slow to apply on a genomic scale. RESULTS: We present an algorithm that efficiently scans a DNA database for short (approximately 20-30 base) sequences that will bind to a query sequence. We use a filtering approach, in which a series of increasingly stringent filters is applied to a set of candidate k-mers. The k-mers that pass all filters are then located in the sequence database using a precomputed index, and an accurate model of DNA binding stability is applied to the sequence surrounding each of the k-mer occurrences. This approach reduces the time to identify all binding partners for a given DNA sequence in human genomic DNA by approximately three orders of magnitude, from two days for the ENCODE regions to less than one minute for typical queries. Our approach is scalable to large DNA sequences. Our method can scan the human genome for medium strength binding sites to a candidate PCR primer in an average of 34.5 minutes. AVAILABILITY: Software implementing the algorithms described here is available at http://noble.gs.washington.edu/proj/dna-binding.     

Insertion of an intermediate repetitive sequence into a sea urchin histone-gene spacer

Summary A common polymorphism of the early embryonic histone-gene repeat ofStrongylocentrotus purpuratus is a 195-bp insertion within the H4-H2B spacer. The sequence, found as an insert in histone-gene repeats of 6 of 22 individuals screened, is also found at approximately 50 sites elsewhere in the genome of every individual. We compare the sequences of the histone-gene spacers that do and do not contain the insert. The insert is found not to have transposon-like features, and no sequence in the original spacer has been duplicated to flank the insert. There is, however, a hexanucleotide sequence that is repeated three times at one end of the insert, and the element has inserted between direct repeats of 5 bp that were present in the original spacer. One of the copies found outside the histone gene cluster was cloned and sequenced and is compared with the insert. Again, no transposon-like features are evident. Regions flanking the homologous sequence in this clone were used as hybridization probes in whole-genome blots. Results indicate that the 195-bp sequence insert is itself embedded within a larger element that is repeated within the genome. Therefore, only a portion of a larger repetitive sequence has integrated into the histone-gene spacer. The sequence features of the insert, although not typical of mobile elements, may be representative of other illegitimate recombination events.     

Herpes simplex virus type 1 variant a sequence generated by recombination and breakage of the a sequence in defined regions, including the one involved in recombination.

A herpes simplex virus type 1 clone, GN29, having exclusively the variant a sequence was isolated. This a sequence was composed of unique (U) and directly repeated (DR) elements DR1, Ub, (DR2)14, Ucd, Ubd, (DR2)5, DR4n2, and Uc and was assumed to be generated by recombination between sites in Ub and Uc. Unusual DNA fragments containing parts of the a sequence, present in the DNA preparations of GN29, were molecularly cloned. Almost all termini of the cloned unusual DNA fragments were situated in defined regions assumed to be recombinogenic: (i) a site in the inverted repeat of the L component, (ii) DR1, (iii) DR2, (iv) the DR4 stretch, and (v) the novel recombination stretch in the variant a sequence of GN29. The termini of unusual DNA fragments, possibly produced by strand breaks, can serve as free DNA ends to initiate recombination of the a sequence. These results support the model of double-strand-break repair for recombination of the a sequence. Sequence-specific enhancement of the recombination of the a sequence probably depends on the presence of recombinogenic elements apt to break, such as DR2 repeats and the DR4 stretch.     

Complete sequence of three alpha-tubulin cDNAs in Chinese hamster ovary cells: each encodes a distinct alpha-tubulin isoprotein.

The genome of Chinese hamster ovary (CHO) cells contains a complex family of approximately 16 alpha-tubulin genes, many of which may be pseudogenes. We present here the complete cDNA sequences of three expressed alpha-tubulin genes; one of these genes has been identified only in CHO cells. The noncoding regions of these three CHO alpha-tubulin genes differed significantly, but their coding regions were highly conserved. Nevertheless, we observed differences in the predicted amino acid sequences for the three genes. A comparison of the CHO alpha-tubulin sequences with all of the sequences available for mammals allowed assignment of the alpha-tubulin genes to three classes. The proteins encoded by the members of two of these classes showed no class-specific amino acids among the mammalian species examined. The gene belonging to the third class encoded an isoprotein which was clearly distinct, and members of this class may play a unique role in vivo. Sequencing of the three alpha-tubulin genes was also undertaken in CMR795, a colcemid-resistant clonal CHO cell line which has previously been shown to have structural and functional alterations in its tubulin proteins. We found differences in the tubulin nucleotide sequence compared with the parental line; however, no differences in the alpha-tubulin proteins encoded in the two cell lines were observed.     

Carbopeptides: chemoselective ligation of peptide aldehydes to an aminooxy-functionalized D-galactose template.

Multifunctional, topological template molecules such as linear and cyclic peptides have been used for the attachment of peptide strands to form novel protein models of, for example, 4-alpha-helix bundles. The concept of carbohydrates as templates for de novo design of potential protein models has been previously described and these novel chimeric compounds were termed carbopeptides. Here, a second generation strategy in which carbopeptides are synthesized by chemoselective ligation of a peptide aldehyde to an aminooxy-functionalized alpha-D-galactopyranoside is described. This template was prepared by per-O-acylation of methyl alpha-D-galactopyranoside with N,N-Boc2-aminooxyacetic acid to form a tetra-functionalized template, followed by treatment with TFA-CH2Cl2 to release the aminooxy functionality. The peptide aldehydes Fmoc-Ser-Gly-Gly-H and H-Ala-Leu-Ala-Lys-Leu-Gly-Gly-H were synthesized by a BAL strategy. Four identical copies of peptide aldehyde were smoothly attached to the template by chemoselective ligation to form a 2.1 and a 2.9 kDa carbopeptide, respectively.     

Investigations into the ability of an oblique alpha-helical template to provide the basis for design of an antimicrobial anionic amphiphilic peptide

AP1 (GEQGALAQFGEWL) was shown by theoretical analysis to be an anionic oblique-orientated alpha-helix former. The peptide exhibited a monolayer surface area of 1.42 nm(2), implying possession of alpha-helical structure at an air/water interface, and Fourier transform infrared spectroscopy (FTIR) showed the peptide to be alpha-helical (100%) in the presence of vesicle mimics of Escherichia coli membranes. FTIR lipid-phase transition analysis showed the peptide to induce large decreases in the fluidity of these E. coli membrane mimics, and Langmuir-Blodgett trough analysis found the peptide to induce large surface pressure changes in monolayer mimics of E. coli membranes (4.6 mN.m(-1)). Analysis of compression isotherms based on mixing enthalpy (DeltaH) and the Gibbs free energy of mixing (DeltaG(Mix)) predicted that these monolayers were thermodynamically stable (DeltaH and DeltaG(Mix) each negative) but were destabilized by the presence of the peptide (DeltaH and DeltaG(Mix) each positive). The peptide was found to have a minimum lethal concentration of 3 mm against E. coli and was seen to cause lysis of erythrocytes at 5 mm. In combination, these data clearly show that AP1 functions as an anionic alpha-helical antimicrobial peptide and suggest that both its tilted peptide characteristics and the composition of its target membrane are important determinants of its efficacy of action.     

Analysis of the regions flanking the human insulin gene and sequence of an Alu family member.

The regions around the human insulin gene have been studied by heteroduplex, hybridization and sequence analysis. These studies indicated that there is a region of heterogeneous length located approximately 700 bp before the 5' end of the gene; and that the 19 kb of cloned DNA which includes the 1430 bp insulin gene as well as 5650 bp before and 11,500 bp after the gene is single copy sequence except for 500 bp located 6000 bp from the 3' end of the gene. This 500 bp segment contains a member of the Alu family of dispersed middle repetitive sequences as well as another less highly repeated homopolymeric segment. The sequence of this region was determined. This Alu repeat is bordered by 19 bp direct repeats and also contains an 83 bp sequence which is present twice. The regions flanking the human and rat I insulin genes were compared by heteroduplex analysis to localize homologous sequences in the flanking regions which could be involved in the regulation of insulin biosynthesis. The homology between the two genes is restricted to the region encoding preproinsulin and a short region of approximately 60 bp flanking the 5' side of the genes.