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R F Doolittle

Publications and source records attributed to R F Doolittle.

196 records · Page 11Linked to original sources
PubMed1969

Identification of the polypeptide chains involved in the cross-linking of fibrin.

The stabilization of fibrin clots by activated factor XIII involves two different sets of cross-linked chains. In one case (type I) two gamma-chains are linked to each other, indicating that gamma-chains have both donor (suitable lysyl-) and acceptor (suitable glutaminyl-) functions. A second system (type II) consists of a gamma-chain linked to an alpha-chain. Experiments with a substitute donor (glycine ethylester) indicate that only gamma-chains have enzyme-accessible acceptor sites, suggesting that alpha-chain participation is limited to lysyl side chains. A model molecular arrangement for fibrin has been suggested which accommodates all the data.

Amino Acids↗
PubMed1967

Light chains of rabbit immunoglobulin: assignment to the kappa class.

Normal rabbit gamma globulin was reduced under conditions presumed to break only interchain disulfide bridges, and the reduiced product was then blocked with C(14)-iodoacetamide. The light chains were separated from the heavy chains and subjected to peptic digestion. Two radioactive peptides were recovered from the digest. The peptides are apparently overlapping and represent the carboxyterminuis. Comparison of this region in the rabbit light chains with the corresponding amino acid sequences in various mouse and human light chains indicates that the rabbit light chains are of the (K)-class.

Amino Acid Sequence↗
PubMed1966

Evolution of immunoglobulin polypeptide chains: carboxy-terminal of an IgM heavy chain.

The dipeptide sequence at the carboxy-terminal of a heavy (micro) chain from a human macroglobulin ( IgM) is tyrosylcysteine, although the reverse sequence, cysteinyltyrosine, has not been rigorously excluded. The presence of cysteine at the carboxy-terminal was predicted from a recognition of the chemical homologies among the polypeptide chains of immunoglobulins, and their probable evolutionary origin.

Amino Acid Sequence↗
PubMed1966

Antibody active sites and immunoglobulin molecules.

In order to obtain detailed information about the relationship between structure and function in antibody molecules, a method called affinity labeling has been devised to attach chemical labels specifically to amino acid residues in the active sites of antibody molecules. With antibodies to three different haptens, highly specific labeling of the active sites has been achieved. Tyrosine residues on both heavy and light polypeptide chains have been labeled in a molar ratio close to 2:1, and labels on the two chains are equally specific to the active sites. Peptide fragmentation studies of the labeled chains of one antibody system have shown that: (i) within 25 amino acid residues of the labeled tyrosine on either chain, substantial chemical heterogeneity exists among different antibody molecules of the same specificity; and (ii) the labeled peptide fragments from both chains are very similar in physicochemical characteristics, including average size, heterogeneity, and unusual hydrophobicity. These experimental results have led us to the view that a particular region of the heavy chain and a particular region of the light chain are utilized to construct the active sites of the three different antibodies, differences in specificity arising from chemical perturbations in these two regions. Correlated structural studies of affinity-labeled antibodies and of the homogeneous light chains (Bence Jones proteins) and heavy chains produced in multiple myeloma may permit the identification of these special active-site regions. The view that active sites of different specificity are chemical perturbations of a particular region of the antibody molecule has a possible close analogue in enzyme systems, particularly among the esterases. The marked chemical similarities we have observed between the active site regions of heavy and light chains indicate to us that chemical homologies, but not identities, exist between the chains. This is reinforced by recently obtained amino acid sequence data which reveal homologies between the two chains near their carboxyl-terminals. These results indicate that the structural genes which code for the synthesis of heavy and light chains are related, presumably having arisen from some common ancestral gene during evolution. This conclusion strongly suggests that both heavy and light chains determine antibody specificity, and has important implications for the still-unknow mechanisms of antibody biosynthesis.

Amino Acid Sequence↗
PubMedDate not supplied

More homologies among the vertebrate plasma proteins.

Many of the proteins of vertebrate blood plasma share common ancestry. As more sequences are reported, the network of relationships continues to expand in unexpected directions. Computer analysis now reveals that a minor plasma protein of unknown function, gamma-trace protein, is related to the kininogen family. Some other possible relationships have been uncovered also, including a resemblance between the histidine-rich hinge regions of high molecular weight kininogen and hemopexin and between Factor VIII and Von Willebrand Factor.

Animals↗
PubMedDate not supplied

Aligning amino acid sequences: comparison of commonly used methods.

We examined two extensive families of protein sequences using four different alignment schemes that employ various degrees of "weighting" in order to determine which approach is most sensitive in establishing relationships. All alignments used a similarity approach based on a general algorithm devised by Needleman and Wunsch. The approaches included a simple program, UM (unitary matrix), whereby only identities are scored; a scheme in which the genetic code is used as a basis for weighting (GC); another that employs a matrix based on structural similarity of amino acids taken together with the genetic basis of mutation (SG); and a fourth that uses the empirical log-odds matrix (LOM) developed by Dayhoff on the basis of observed amino acid replacements. The two sequence families examined were (a) nine different globins and (b) nine different tyrosine kinase-like proteins. It was assumed a priori that all members of a family share common ancestry. In cases where two sequences were more than 30% identical, alignments by all four methods were almost always the same. In cases where the percentage identity was less than 20%, however, there were often significant differences in the alignments. On the average, the Dayhoff LOM approach was the most effective in verifying distant relationships, as judged by an empirical "jumbling test." This was not universally the case, however, and in some instances the simple UM was actually as good or better. Trees constructed on the basis of the various alignments differed with regard to their limb lengths, but had essentially the same branching orders. We suggest some reasons for the different effectivenesses of the four approaches in the two different sequence settings, and offer some rules of thumb for assessing the significance of sequence relationships.

Amino Acid Sequence↗
PubMedDate not supplied

Computer-based characterization of epidermal growth factor precursor.

The cDNA sequence of the precursor of mouse epidermal growth factor (EGFP) has recently been reported by two groups, both of whom noted the presence of repeated similar segments, each about 40 residues long. One of these repeat units overlaps with the sequence of epidermal growth factor itself. The sequence of epidermal growth factor has been reported to be similar to that of pancreatic secretory trypsin inhibitor (PSTI) and a somewhat better match has been found with part of the sequence of bovine factor X, one of the blood coagulating factors. We report here that there is an even stronger similarity between the sequences of some of the repeat units of epidermal growth factor precursor and certain segments in factor X. This sequence similarity is also apparent in comparisons with other blood coagulation factors. On the basis of these sequence comparisons we suggest a scheme for the evolution of the epidermal growth factor precursor. We have also identified certain structural features in the precursor sequence that bear on the way in which the mature epidermal growth factor is generated.

Amino Acid Sequence↗
PubMedDate not supplied

A relationship between the yeast cell cycle genes CDC4 and CDC36 and the ets sequence of oncogenic virus E26.

We report here significant primary sequence homology among the predicted translational products of three genes: CDC4 , CDC36 and ets. CDC4 and CDC36 are Saccharomyces cerevisiae cell division cycle genes, while ets is a transformation-specific sequence of avian erythroblastosis virus E26. The deduced primary structures of the three gene products were compared by computer to a large data base of known and predicted protein sequences. The search revealed 22.0-25.5% identity over regions of 140-206 codons, respectively between the different pairwise combinations. For these particular sequences, these identity scores fall 3.4-4.0 standard deviations above the empirically-determined mean values of fortuitous similarity. S. cerevisiae calls require CDC36 and CDC4 in order to complete two early events in the cell cycle: execution of start ( CDC36 ) and spindle pole body separation ( CDC4 ). In virus E26, the ets sequence is linked in frame with delta gag and mybE in the tripartite structure 5'-delta gag- mybE -ets-3', comprising the E26 transforming oncogene. The homologies described here suggest that the biochemical functions or regulation of the CDC4 , CDC36 and ets products may be related.

Animals↗
PubMedDate not supplied

Six unidentified reading frames of human mitochondrial DNA encode components of the respiratory-chain NADH dehydrogenase.

The products of six unidentified reading frames of human mitochondrial DNA are precipitated from a mitochondrial lysate by antibodies against highly purified native beef heart NADH-ubiquinone oxidoreductase (complex I). These products are enriched greatly in a human submitochondrial fraction enriched in NADH-Q1 and NADH-K3Fe(CN)6 oxidoreductase activities. We conclude that the six reading frames encode components of the respiratory-chain NADH dehydrogenase.

Animals↗
PubMedDate not supplied

Domainal evolution of a prokaryotic DNA repair protein and its relationship to active-transport proteins.

The ABC excision nuclease of Escherichia coli is an ATP-dependent DNA repair enzyme composed of three protein subunits, UvrA, UvrB and UvrC. The DNA sequences of all three genes have been reported. UvrA, the component that binds directly to the DNA, and UvrB, which attaches itself to the UvrA-DNA complex, both contain consensus sequences though to be diagnostic of ATP-binding sites, although the UvrC sequence does not. We now report that a computer analysis of the UvrA sequence has revealed an unusual series of internal duplications centering around putative metal-binding sites which may be involved in the interaction with DNA. We also find a strong evolutionary relationship to a family of prokaryotic membrane-associated active-transport proteins.

Amino Acid Sequence↗
PubMedDate not supplied

Evidence for the Heterolobosea from phylogenetic analysis of genes encoding glyceraldehyde-3-phosphate dehydrogenase.

The phylogenetic relationships between major slime mould groups and the identification of their unicellular relatives has been a subject of controversy for many years. Traditionally, it has been assumed that two slime mould groups, the acrasids and the dictyostelids were related by virtue of their cellular slime mould habit; a view still endorsed by at least one current classification scheme. However, a decade ago, on the basis of detailed ultrastructural resemblances it was proposed that acrasids of the family Acrasidae were not relatives of other slime moulds but instead related to a group of mostly free-living unicellular amoebae, the Schizopyrenida. The class Heterolobosea was created to contain these organisms and has since figured in many discussions of protist evolution. We sought to test the validity of Heterolobosea by characterizing homologs of the highly conserved glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from an acrasid, Acrasis rosea; a dictyostelid, Dictyostelium discoideum; and the schizopyrenid Naegleria andersoni. Phylogenetic analysis of these and other GAPDH sequences, using maximum parsimony, neighbour-joining distance and maximum likelihood methods strongly supports the Heterolobosea hypothesis and discredits the concept of a cellular slime mould grouping. Moreover, all of our analyses place Dictyostelium discoideum as a relatively recently originating lineage, most closely related to the Metazoa, similar to other recently published phylogenies of protein-coding genes. However, GAPDH phylogenies do not show robust branching orders for most of the relationships between major groups. We propose that several of the incongruencies observed between GAPDH and other molecular phylogenies are artifacts resulting from substitutional saturation of this enzyme.

Amino Acid Sequence↗