P.D. Baas

Islet amyloid polypeptide: structure and upstream sequences of the IAPP gene in rat and man.

Islet amyloid polypeptide (IAPP) or amylin is a pancreatic islet hormone which was first found in amyloid in insulinomas and in pancreases of patients with type 2 diabetes. In rat a similar polypeptide occurs; however, pancreatic amyloid in this species has not been described. Here we report the structure of the rat and human IAPP gene. Both consist of three exons and two introns which are very similar. The upstream sequence of the rat IAPP gene contains a TATA-box, a CCAAT-sequence and a GT-element, whereas the upstream sequence of the human IAPP gene contains a TATA-box and a rat insulin enhancer-like sequence. This suggests that the rat and human IAPP gene may be controlled differently at the transcriptional level.

Arthrobacter luteus restriction endonuclease cleavage map of ∅X174 RF DNA

Abstract 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).

Deregulation of alternative processing of Calcitonin/CGRP-I pre-mRNA by a single point mutation.

The Calcitonin/CGRP-I (CALC-I) gene was one of the first examples of a cellular gene exhibiting alternative, tissue-specific processing of its primary transcript. Calcitonin (CT) mRNA is the predominant product in thyroid C-cells, whereas CGRP-I (Calcitonin Gene Related Peptide-I) mRNA is the main product in neurons of the central and peripheral nervous systems. Investigating the molecular mechanism underlying the alternative processing events, we have demonstrated that the CT-specific splice acceptor site is an intrinsical weak site due to usage of a uridine branch acceptor. The data presented in this report show that a single point mutation changing the uridine branch acceptor into a commonly preferred adenosine residue results in the predominant production of CT mRNA in otherwise CGRP-I mRNA-producing F9 cells. The results of the experiments implicate that the low efficiency of CT splicing, caused by usage of a uridine branch acceptor, allows the production of CGRP-I mRNA in neural cells.

Termination and reinitiation signals of bacteriophage φX174 rolling circle DNA replication

The nucleotide sequence requirements for termination and reinitiation of rolling circle DNA replication within the 30-bp phi X174 origin region were studied. Plasmids were constructed which contained a complete and a partial phi X174 origin region in the same orientation. The partial origin consisted of the first 16, 24, 25, 26, 27, or 28 bp of the origin region. Plasmids harboring a complete origin region are subject to rolling circle DNA replication and packaging of single-stranded plasmid DNA into phage coats in phi X174 or G4 phage infected cells. The plasmids with a complete and partial origin region were tested in these in vivo transduction systems. The results lead to the following conclusions: The phi X174 and G4 in vivo transduction systems are useful in studying termination and reinitiation of rolling circle DNA replication. The first 24 bp of the origin region are sufficient for termination of a round of rolling circle DNA replication coupled to DNA packaging. The first 16 bp, however, are not recognized as a termination signal. Reinitiation of rolling circle DNA replication coupled to DNA packaging on a partial origin region occurs with low frequency.

The Role of Small GTPases in Signal Transduction

Small GTPases are proteins of about 21kD that cycle between an active GTP-bound state and an inactive GDP-bound state. They can be considered as molecular switches in a variety of regulatory processes, including signal transduction (the Ras family), cytoskeletal reorganisation (Rac and Rho family) and vesicle transport (Rab family) (Figure 1).

STUDIES ON THE ORIGIN OF ∅X RF DNA REPLICATION

ABSTRACT This paper describes our current research on the subject of which nucleotide sequence in bacteriophage ∅X174 DNA is essential for a functional origin in the process of DNA replication. Results of three different approaches are reported.

Replication of ϕ X174 RF DNA In Vivo

The first clue toward understanding the mode of ϕ X174 DNA replication was the discovery by Sinsheimer et al. (1962) of an intracellular double-stranded form (replicative form [RF]) of bacteriophage ϕ X DNA. Until that time it was a puzzle how a single-stranded DNA molecule could replicate. The mediation of a double-stranded form seemed to solve this problem, and a few years later Denhardt and Sinsheimer (1965a) and Stone (1967) were able to show that ϕ X RF DNA replicates in a semiconservative manner. Three stages of DNA synthesis can be distinguished during the ϕ X life cycle (for reviews see Sinsheimer 1968; Denhardt 1975, 1977; Dressler et al., this volume). The first RF in the ϕ X-infected Escherichia coli C host derives from the de novo synthesis of a complementary strand on the infecting viral single-stranded (SS) DNA (SS→RF), which is accomplished by preexisting host enzymes. This parental duplex RF DNA then replicates to form a pool of some 20 RF molecules (RF→RF) at 15 minutes after infection; this requires several host enzymes as well as one phage-encoded enzyme, the gene- A protein. In the final stage of the ϕ X life cycle, SS DNA of the progeny virus is derived from RF DNA by asymmetric synthesis (RF→SS). This paper reviews the second stage of ϕ X DNA replication (RF→RF) in vivo and is for the most part limited to labeling studies and to analyses of replicating intermediates by biochemical and electron microscopic methods. Other approaches, namely, the effect of blocking host-cell functions by mutation and studies...