Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase.

5'-Noncoding sequences have been compiled from 699 vertebrate mRNAs. (GCC) GCCA/GCCATGG emerges as the consensus sequence for initiation of translation in vertebrates. The most highly conserved position in that motif is the purine in position -3 (three nucleotides upstream from the ATG codon); 97% of vertebrate mRNAs have a purine, most often A, in that position. The periodical occurrence of G (in positions -3, -6, -9) is discussed. Upstream ATG codons occur in fewer than 10% of vertebrate mRNAs-at-large; a notable exception are oncogene transcripts, two-thirds of which have ATG codons preceding the start of the major open reading frame. The leader sequences of most vertebrate mRNAs fall in the size range of 20 to 100 nucleotides. The significance of shorter and longer 5'-noncoding sequences is discussed.

An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs

Activated protein C (APC) is a serine protease with potent anticoagulant properties, which is formed in blood on the endothelium from an inactive precursor During normal haemostasis, APC limits clot formation by proteolytic inactivation of factors Va and VIIIa (ref 2) To do this efficiently the enzyme needs a nonenzymatic cofactor, protein S (ref 3) Recently it was found that the anticoagulant response to APC (APC resistance) was very weak in the plasma of 21% of unselected consecutive patients with thrombosis and about 50% of selected patients with a personal or family history of thrombosis; moreover, 5% of healthy individuals show APC resistance, which is associated with a sevenfold increase in the risk for deep vein thrombosis Here we demonstrate that the phenotype of APC resistance is associated with heterozygosity or homozygosity for a single point mutation in the factor V gene (at nucleotide position 1,691, G-->A substitution) which predicts the synthesis of a factor V molecule (FV Q506, or FV Leiden) that is not properly inactivated by APC The allelic frequency of the mutation in the Dutch population is approximately 2% and is at least tenfold higher than that of all other known genetic risk factors for thrombosis (protein C (ref 8), protein S (ref 9), antithrombin10 deficiency) together

/pdf/mutation-in-blood-coagulation-factor-v-associated-with-33avp1ullq.pdf

Mutation in blood coagulation factor V associated with resistance to activated protein C

Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction

The small (40S) subunit of eukaryotic ribosomes is believed to bind initially at the capped 5'-end of messenger RNA and then migrate, stopping at the first AUG codon in a favorable context for initiating translation. The first-AUG rule is not absolute, but there are rules for breaking the rule. Some anomalous observations that seemed to contradict the scanning mechanism now appear to be artifacts. A few genuine anomalies remain unexplained.

/pdf/the-scanning-model-for-translation-an-update-52r2in8l71.pdf

The scanning model for translation: an update.

We have isolated and sequenced several cDNAs derived from the sea squirt Ciona intestinalis that encode vitamin K-dependent proteins. Four of these encode γ-carboxyglutamic acid (Gla) domain-containing proteins, which we have named Ci-Gla1 through Ci-Gla4. Two additional cDNAs encode the apparent orthologs of γ-glutamyl carboxylase and vitamin K epoxide reductase. Ci-Gla1 undergoes γ-glutamyl carboxylation when expressed in CHO cells and is homologous to Gla-RTK, a putative receptor tyrosine kinase previously identified in a related ascidian. The remaining three Gla domain proteins are similar to proteins that participate in fundamental developmental processes, complement regulation, and blood coagulation. These proteins are generally expressed at low levels throughout development and exhibit either relatively constant expression (Ci-Gla1, γ-glutamyl carboxylase, and vitamin K epoxide reductase) or spatiotemporal regulation (Ci-Gla2, -3, and -4). These results demonstrate the evolutionary emergence of the vitamin K-dependent Gla domain before the divergence of vertebrates and urochordates and suggest novel functions for Gla domain proteins distinct from their roles in vertebrate hemostasis. In addition, these findings highlight the usefulness of C. intestinalis as a model organism for investigating vitamin K-dependent physiological phenomena, which may be conserved among the chordate subphyla.

https://www.pnas.org/content/pnas/103/43/15794.full.pdf

Vitamin K-dependent proteins in Ciona intestinalis, a basal chordate lacking a blood coagulation cascade.

Factor VIII is present in plasma in a precursor or inactive form. When bovine factor VIII that has been purified approximately 10,000-fold is incubated with thrombin, an activated product is formed which participates in the conversion of factor X to factor Xa in the presence of factor IXa, calcium ions, and phospholipid. This activated product, which has been tentatively identified as activated factor XIII, was stable when formed in the presence of 0.25M CaCl2 but was rapidly inactivated in the absence of CaCl2. It was inhibited by diisopropyl phosphorofluoridate and antithrombin III, suggesting that it is a serine enzyme. The exact role of this serine enzyme in the intrinsic pathway of coagulation remains to be established.

Formation of a serine enzyme in the presence of bovine factor VIII (antihemophilic factor) and thrombin.

Action of Carboxypeptidase on β-Lactoglobulin

Transcriptional Regulation of the Gene Coding for Human Protein C

Baby hamster kidney cells were transformed with DNA sequences derived from the gene for human angiogenin. Expression was under the transcriptional control of the inducible mouse metallothionein 1 promoter. Recombinant angiogenin was purified and shown to be chemically, biologically, and enzymatically indistinguishable from the natural product. The large-scale production of recombinant angiogenin achieved should facilitate detailed studies into the structure-function relationships of this potent angiogenic molecule.

Earl W. Davie

Papers

Vitamin K-dependent proteins in Ciona intestinalis, a basal chordate lacking a blood coagulation cascade.

Formation of a serine enzyme in the presence of bovine factor VIII (antihemophilic factor) and thrombin.

Action of Carboxypeptidase on β-Lactoglobulin

Transcriptional Regulation of the Gene Coding for Human Protein C

Expression of human angiogenin in cultured baby hamster kidney cells.