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R F Doolittle

Publications and source records attributed to R F Doolittle.

At least 109 records · Page 6Linked to original sources
PubMed1986

A method for the simultaneous alignment of three or more amino acid sequences.

We describe an algorithm for the concurrent comparison of three or more amino acid sequences. The basis of the approach is a progressive evaluation of selected segments from each sequence. Only a small subset of all possible segments from each sequence is compared, and a minimum of information is retained for the trace-back of the alignment. As a result, this method has the advantage of being both rapid and minimally consumptive of computer memory when constructing an alignment. This being the case, there are no practical limits on the length of sequences that may be aligned. A computer program for the alignment of three sequences is described, and this method is compared with two three-sequence extensions of the Needleman and Wunsch variety, including a recently published approach. In addition, we have made simultaneous alignments of sets of four and five sequences with this selected-segment method.

Amino Acid Sequence↗
PubMed1986

Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes.

A computer analysis of the amino acid sequences from the putative gene products of retroviral pol genes has revealed a 150-residue segment that is homologous with the ribonuclease H of Escherichia coli. The segment occurs at the carboxyl terminus of the region assigned to the 90-kDa reverse transcriptase polypeptide. In contrast, a section nearer the amino terminus of this sequence can be aligned with nonretroviral polymerases. The order of activities in the pol gene is thus: polymerase-ribonuclease-endonuclease. On another note, all retroviral endonuclease sequences contain a consensus zinc-binding "finger." This should not be confused with the well-known zinc requirement of reverse transcriptases.

Amino Acid Sequence↗
PubMed1986

Identification of the polypeptides encoded in the unassigned reading frames 2, 4, 4L, and 5 of human mitochondrial DNA.

In previous work, antibodies prepared against chemically synthesized peptides predicted from the DNA sequence were used to identify the polypeptides encoded in three of the eight unassigned reading frames (URFs) of human mitochondrial DNA (mtDNA). In the present study, this approach has been extended to other human mtDNA URFs. In particular, antibodies directed against the NH2-terminal octapeptide of the putative URF2 product specifically precipitated component 11 of the HeLa cell mitochondrial translation products, the reaction being inhibited by the specific peptide. Similarly, antibodies directed against the COOH-terminal nonapeptide of the putative URF4 product reacted specifically with components 4 and 5, and antibodies against a COOH-terminal heptapeptide of the presumptive URF4L product reacted specifically with component 26. Antibodies against the NH2-terminal heptapeptide of the putative product of URF5 reacted with component 1, but only to a marginal extent; however, the results of a trypsin fingerprinting analysis of component 1 point strongly to this component as being the authentic product of URF5. The polypeptide assignments to the mtDNA URFs analyzed here are supported by the relative electrophoretic mobilities of proteins 11, 4-5, 26, and 1, which are those expected for the molecular weights predicted from the DNA sequence for the products of URF2, URF4, URF4L, and URF5, respectively. With the present assignment, seven of the eight human mtDNA URFs have been shown to be expressed in HeLa cells.

Antibodies↗
PubMed1986

Repetitive segmental structure of the transducin beta subunit: homology with the CDC4 gene and identification of related mRNAs.

Retinal transducin, a guanine nucleotide regulatory protein (referred to as a G protein) that activates a cGMP phosphodiesterase in photoreceptor cells, is comprised of three subunits. We have identified and analyzed cDNA clones of the bovine transducin beta subunit that may be highly conserved or identical to that in other G proteins. From the cDNA nucleotide sequence of the entire coding region, the primary structure of a 340-amino acid protein was deduced. The encoded beta subunit has a Mr of 37,375 and is comprised of repetitive homologous segments arranged in tandem. Furthermore, significant homology in primary structure and segmental sequence exists between the beta subunit and the yeast CDC4 gene product. The Mr 37,375 beta subunit polypeptide is encoded by a 2.9-kilobase (kb) mRNA. However, there exists in retina other beta-related mRNAs that are divergent from the 2.9-kb mRNA on the basis of oligonucleotide and primer-extended probe hybridizations. All mammalian tissues and clonal cell lines that have been examined contain at least two beta-related mRNAs, usually 1.8 and 2.9 kb in length. These results suggest that the mRNAs are the processed products of a small number of closely related genes or of a single highly complex beta gene.

Amino Acid Sequence↗
PubMed1985

Lamprey fibrinogen gamma chain: cloning, cDNA sequencing, and general characterization.

A cDNA library from lamprey liver was constructed in pBR322 and screened with a synthetic mixed oligonucleotide probe, the sequence of which was based on a partial amino acid sequence of the lamprey fibrinogen gamma chain determined by conventional procedures. Among the positive clones was one containing a 600-base insert that covered the carboxy-terminal third of the chain and another with a 1950-base insert that stretched more than full length. The two inserts were sequenced by the Maxam-Gilbert procedure. The DNA sequencing was corroborated by reference to the amino acid sequences of five cyanogen bromide peptides that compose the carboxy-terminal 130 amino acids, as well as to a number of tryptic peptides from elsewhere in the molecule. The clone with the smaller insert (6G) contained 594 nucleotides (not counting G and C tails), 435 of which are coding and correspond to residues 264-408 of the gamma chain. The remaining 159 nucleotides included the terminator codon followed by a noncoding segment. The larger clone (2E) coded for 408 amino acids that could be readily aligned with the 411-residue human gamma chain. A 24-residue signal peptide adjacent to the proposed amino terminal was also inferred. The amino acid sequence of the fibrinogen gamma chain has been differentially conserved during evolution, the lamprey and human sequences being more than 70% identical in certain key regions but dropping to less than 25% in other sections, including the segment thought to be a part of the "coiled coils". Overall, the resemblance amounts to 50% identity. Of the 10 cysteines found in mammalian chains, 9 are at identical positions, but the tenth, which in mammalian fibrinogens is a part of the interdimeric bridging, is absent in the lamprey.

Amino Acid Sequence↗
PubMed1985

Determination of the relative positions of amino acids by partial specific cleavages of end-labeled proteins.

We have developed a new method for obtaining information about protein sequences that uses an approach analogous to that used to determine DNA sequences. In essence, three steps are involved. First, a detectable label is attached exclusively to the amino terminus of a polypeptide. Next, the labeled chain is subjected to partial specific cleavage in a way that produces roughly equimolar amounts of fragments of different sizes. Cleavages for methionine, tryptophan, arginine, aspartyl-proline bonds, and asparaginyl-glycine bonds have been employed. Lastly, the labeled fragments are separated according to size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The distribution of target amino acids along the polypeptide chain can be deduced from the specific pattern of labeled bands by reading the "ladder" in the same way that DNA sequencing gels are read. Although the method can be conducted with a radioactive label, we have chosen to use a fluorescent label. We have applied the method successfully to the three subunit chains of two different fibrinogens.

Amino Acid Sequence↗
PubMed1985

Proteins.

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Amino Acid Sequence↗
PubMed1984

Structure and expression of a complementary DNA for the nuclear coded precursor of human mitochondrial ornithine transcarbamylase.

Most mitochondrial proteins are encoded in the nucleus and are translated on free cytoplasmic ribosomes as larger precursors containing amino-terminal "leader" sequences, which are removed after the precursors are taken up by mitochondria. We have deduced the complete primary structure of the precursor of a human mitochondrial matrix enzyme, ornithine transcarbamylase (OTC), from the nucleotide sequence of cloned complementary DNA. The amino-terminal leader peptide of OTC is 32 amino acids in length and contains four arginines but no acidic residues. Cleavage of the leader peptide from the "mature" protein occurs between glutamine and asparagine residues. The sequence of mature human OTC resembles that of the subunits of both OTC and aspartate transcarbamylase from Escherichia coli. The biological activity of the cloned OTC complementary DNA was tested by joining it with SV40 (an animal virus) regulatory elements and transfecting cultured HeLa cells, which do not normally express OTC. Both the precursor and mature forms of the OTC subunit were identified; in stable transformants, enzymatic activity was also detected.

Amino Acid Sequence↗
PubMed1983

Angiotensinogen is related to the antitrypsin-antithrombin-ovalbumin family.

The recently reported amino acid sequence of rat angiotensinogen was subjected to a computer-assisted search for homology with known sequences stored in a data bank and found to be significantly related to that of plasma alpha 1-antitrypsin, itself a member of a family that includes antithrombin III and ovalbumin. An alignment of the four sequences shows indisputably the common ancestry of all four proteins.

Amino Acid Sequence↗
PubMed1983

Simian sarcoma virus onc gene, v-sis, is derived from the gene (or genes) encoding a platelet-derived growth factor.

The transforming protein of a primate sarcoma virus and a platelet-derived growth factor are derived from the same or closely related cellular genes. This conclusion is based on the demonstration of extensive sequence similarity between the transforming protein derived from the simian sarcoma virus onc gene, v-sis, and a human platelet-derived growth factor. The mechanism by which v-sis transforms cells could involve the constitutive expression of a protein with functions similar or identical to those of a factor active transiently during normal cell growth.

Amino Acid Sequence↗
PubMed1983

Interaction of fibrinogen with staphylococcal clumping factor and with platelets.

Fibrinogen, a clottable plasma glycoprotein, participates in cell adhesion phenomena involving prokaryotic cells, e.g. staphylococci, and eukaryotic cell fragments, e.g. platelets. Among the three chains (alpha, beta, gamma) of human fibrinogen, the gamma chain bears the main site recognizing the staphylococcal clumping receptor and human platelet receptor induced by ADP. The platelet receptors are also recognized, albeit less avidly, by a site associated with the alpha chain. The gamma chain site recognizing staphylococcal clumping factor exists on the COOH-terminal segment of this chain encompassing the 15 residues (gamma 397-411) including the COOH-terminal valine. The location of the gamma chain site interacting with the human platelet receptor had been pinpointed to the 27 residue CNBr COOH-terminal segment (gamma 385-411). The results of enzymatic degradation of the 27-residue peptide indicate that the continuity of the last 15 amino acid residues at the COOH-terminal end of the gamma chain of human fibrinogen seems to be essential for its interaction with human platelets. The sequence of the gamma chain interacting with the platelet receptor (gamma 385-411) indicates that this segment is a unique region of fibrinogen endowed with three important functions: cross-linking of fibrin, clumping of staphylococci, and aggregation of platelets. [Note added in proof: Recently we obtained evidence that dodecapeptide gamma 393-411 fully retains platelet receptor recognition site (Kloczewiak et al. 1983. Clin. Res 31:534A.)]

Amino Acid Sequence↗
PubMed1983

Dimeric half-molecules of human fibrinogen are joined through disulfide bonds in an antiparallel orientation.

Human fibrinogen is a dimer composed of two identical halves. Each dimeric half contains three peptide chains (alpha, beta, and gamma) linked by disulfide bonds. The two half-molecules are joined by three disulfide bonds, one between the two alpha-chains (residue alpha-28) and two between the two gamma-chains (residues gamma-8 and gamma-9). In the absence of any difinitive experimental evidence, it has been presumed that the joined halves were aligned in a parallel orientation similar to the situation found in immunoglobulins. We have now determined that the two gamma-chains--hence, the dimeric halves--are connected in an antiparallel manner. A tryptic peptide containing gamma-chain residues 6-14 was isolated as a disulfide-linked dimer from CNBr-treated fragment E. Synthetic peptides corresponding to this sequence were prepared, from which parallel and antiparallel dimers were constructed. During the syntheses, cysteine thiol groups were protected as p-methoxybenzyl and acetamidomethyl sulfides; the peptides were dimerized by selective deprotection and disulfide bond formation. First, the p-methoxybenzyl groups were removed by liquid hydrogen fluoride and the newly exposed thiols oxidized in the presence of potassium ferricyanide. Then the monocystine compound was converted to the double-cystine product by iodolytic cleavage of the acetamidomethyl group with concomitant disulfide bond formation. This selectivity was used to prepare peptide dimers which modeled both parallel and antiparallel arrangements. The antiparallel-oriented synthetic peptide was indistinguishable from the native tryptic peptide as judged by elution from reverse-phase high-performance liquid chromatography and circular dichroism spectroscopy. The parallel-oriented synthetic peptide differed from the native material by both criteria.

Amino Acid Sequence↗