Arthrobacter luteus restriction endonuclease cleavage map of X174 RF DNA

Cleavage of X174 RF DNA with the restriction endonuclease from Arthrobacter luteus (Alu I) produces 23 fragments of approximately 24–1100 base pairs in length. The order of most of these fragments has been established by digestion of Haemophilus influenzae Rd (Hind II) and Haemophilus aegyptius (Hae III) endonuclease fragments of X RF with Alu I and by reciprocal digestions of Alu I fragments with Hind II and Hae III. In this way the Arthrobacter luteus map could be aligned with the Hind II and Hae III cleavage maps of X174 RF DNA of A. S. Lee and R. L. Sinsheimer ((1974) Proc. Nat. Acad. Sci. USA 71, 2882–2886)

Alteration of the ATG start codon of the A* protein of bacteriophage [phi]X174 into an ATT codon yields a viable phage indicating that A* protein is not essential for [phi]X174 reproduction

Bacteriophage X174 gene A encodes two proteins: the gene A protein and the smaller A* protein, which is synthesized from a translational start signal within the A gene in the same reading frame as the gene A protein. The gene A protein is involved in initiation, elongation and termination of rolling circle DNA replication. The role of the A* protein in the life cycle of X174, however, is unknown. Using oligonucleotide-directed mutagenesis a viable Xl74 mutant was constructed in which the ATG start codon of the A* protein was changed into an ATT codon. This mutant, X-4499T, does not synthesize A* protein. The burst size of X-4499T amounted to 50% of that of wild type XI74. This indicates that A* protein, although advantageous for phage reproduction, is not essential during the life cycle of bacteriophage X174

A* protein of bacteriophage [phi]X174 carries an oligonucleotide which it can transfer to the 3-OH of a DNA chain

The bacteriophage φX174 gene A encodes two proteins: gene A protein and A* protein. Purified A* protein acts as a single-stranded, DNA-specific endonuclease which remains covalently attached to the 5′-end of the cleavage site. Incubation of A* protein with the synthetic heptamer CAACTTG or with oligonucleotides which yield this heptamer after cleavage with the A* protein yields oligonucleotides with the sequences CAACTTGAG, CAACTTGAGG and CAACTTGAGGA. This indicates that A* protein carries an oligonucleotide with the sequence -AG, AGG or -AGGA. The oligonucleotide can be transferred to the 3′-end of the heptamer CAACTTG. This suggests that A* protein reacts with a specific DNA sequence in the infected cell. Author Keywords: Bacteriophage φX174; A* protein; DNA—protein complex; Synthetic oligonucleotide; DNA-ligating activit

Amylin-induced in vivo insulin resistance in conscious rats: the liver is more sensitive to amylin than peripheral tissues

Amylin is a polypeptide of 37 amino acids, predominantly synthesized in pancreatic Beta cells. The peptide was suggested to be dysregulated in Type 2 (non-insulin-dependent) diabetes mellitus and it antagonized certain actions of insulin in vitro in rat muscle. This led to speculation that amylin is involved in the pathogenesis of Type 2 diabetes. We have examined the in vivo effects of rat amylin, amidated at the carboxy-terminus, on insulin-mediated carbohydrate metabolism in conscious rats, using the hyperinsulinaemic (±1 nmol/l) euglycaemic (6 mmol/l) clamp technique combined with [3-3H]-glucose infusion. Basal plasma amylin levels were ≤75 pmol/l. Applied amylin levels of 220 ± 75 pmol/l (infusion rate of 12.5 pmol/min) antagonized only the insulin action on liver, resulting in a 100% increase of hepatic glucose output. Amylin levels of 4750 ± 750 pmol/l (infusion rate of 125 pmol/min) induced a 250% increase of insulin-inhibited hepatic glucose output and, in addition, a 30% decrease of insulin-stimulated peripheral glucose uptake. Amylin did not affect: 1) the metabolic clearance rate of insulin, 2) the levels of plasma glucagon, epinephrine, norepinephrine, and corticosterone, 3) in vitro insulin binding and insulin-stimulated receptor autophosphorylation. This suggests that amylin antagonizes insulin action via binding to a yet unknown receptor. In conclusion: amylin causes in vivo insulin resistance and the liver seems the predominant organ regulated by this hormone. The in vivo effects of amylin mimic the pathophysiological abnormalities of insulin action in Type 2 diabetes.

The bond in the bacteriophage øX174 gene A protein-DNA complex is a tyrosyl-5'-phosphate ester

AbstractThe bacteriophage øX174 gene A protein cleaves the viral strand of the double-stranded replicative form (RF) DNA of the phage at a specific site, the origin. It leaves a free 3'-OH at nucleotide 4305 (G) of the øx DNA sequence and binds covalently to the DNA. The nature and position of the covalent bond have been determined using the octadecadesoxyribonucleotide CAACTTG[32P]ATATTAATAAC. This octadecamer, which corresponds to nucleotides 4299–4316 of øX viral DNA, is cleaved by gene A protein. Gene A protein is bound to the labelled phosphate via a tyrosyl residue, indicating that binding occurs to the nucleotide corresponding to 4306 (A) of the øX viral DNA strand.Bacteriophage øX174Gene A proteinDNA replicationProtein-DNA complexSynthetic oligonucleotideTyrosyl-5'-phosphate este

Islet amyloid polypeptide: Identification and chromosomal localization of the human gene

Abstract Islet or insulinoma amyloid polypeptide (IAPP) is a 37 amino acid polypeptide isolated from pancreatic amyloid. Here, we describe the isolation and partial characterization of the human gene encoding IAPP. The DNA sequence predicts that IAPP is excised from a larger precursor protein and that its carboxy-terminus is probably amidated. The predicted normally occurring IAPP is identical to the reported polypeptides isolated from pancreatic amyloid, except for the amidated carboxy-terminus. IAPP specific polyadenylated RNAs of 1.6 kb and 2.1 kb are present in human insulinoma RNA. The human IAPP gene is located on chromosome 12