A.D.M. van Mansfeld

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

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.

Discordance of p53 status in matched primary tumours and metastases in head and neck squamous cell carcinoma patients

To study the use of p53 as a diagnostic tool in head and neck squamous cell carcinoma (HNSCC), we analysed 15 primary tumours (PT) and matched lymph node metastases (LNM) for overexpression and mutations of p53. The primary goal was to study whether differentiation between primary and metastatic disease through their p53 status would be possible. Immunohistochemistry for p53 protein (antibody BP 53-12-1) was performed. Mutations of the p53 gene were detected by exon-specific amplification of DNA (exons 4-9), followed by exon analysis using denaturing gradient gel electrophoresis (DGGE). Mutant exons were sequenced. p53 overexpression was detected in seven (47%) of the PT and in seven (47%) of the LNM. 6 patients (40%) exhibited p53 protein overexpression in both PT and LNM. 2 patients had a different p53 protein expression in each sample. Mutations in the p53 gene were detected in 6 patients (40%) in the PT and in 7 patients (47%) in the LNM. In 2 patients (13%), the same mutation was found in the PT and in the LNM. 9 patients (60%) had a different mutation in each sample. We conclude that a poor correlation exists between p53 protein overexpression and p53 gene mutation in HNSCC. Also, a poor correlation for both detection techniques exists, when PT and LNM are compared. The p53 status may seem to differ between PT and LNM because of polyclonality in the PT. More sensitive detection techniques could be promising.

Construction of viable and lethal mutations in the origin of bacteriophage φX174 using synthetic oligodeoxyribonucleotides

Abstract Six different synthetic deoxyhexadecamers complementary to the origin of bacteriophage φX174, corresponding to nucleotides 4299 to 4314, except for one preselected nucleotide change were used as primers for DNA synthesis on wild-type φX † DNA as a template. DNA synthesis was performed with Escherichia coli DNA polymerase I (Klenow fragment) in the presence of DNA ligase. Heteroduplex RFIV DNA was isolated and, after limited digestion with DNAase I, complementary strands containing the mutant primers were isolated. The biological activity of these complementary strands was assayed in spheroplasts. Spheroplasts were made from E. coli K58 ung − (uracil N -glycosylase) to prevent degradation of the complementary strands caused by uracil incorporation (Baas et al. , 1980 a ). Using (5′- 32 P) end-labeled primers, it was shown that all tested DNA polymerase preparations, including phage T4 DNA polymerase, contained variable amounts of 5′ → 3′ exonuclease activity. This nick translation activity may result in removal of the mutation in the primers, and therefore in isolation of wild-type complementary DNA instead of mutant complementary DNA. Restriction enzyme analysis of completed RFIV DNA showed that the primers can initiate DNA synthesis at more than one place on the φX174 genome. These complications result in a mixed population of complementary strand DNAs synthesized in vitro . Nevertheless, the desired mutants were picked up with high frequency using a selection test that is based on the difference in ultraviolet light sensitivity of homoduplex and heteroduplex φX174 RF DNA. Heteroduplex φX174 RF DNA is two to three times more sensitive to ultraviolet light irradiation than is homoduplex φX174 RF DNA (Baas & Jansz, 1971,1972). Phage DNA derived from single plaque lysates of two of the six mutant complementary strand DNA preparations yielded, after annealing with wild-type complementary strand DNA, heteroduplex DNA with high frequency. DNA sequence analysis in the origin region of RF DNA obtained from these two phage preparations revealed the presence of the expected mutation. RFI DNA of these two origin mutants was nicked by φX174 gene A protein in the same way as wild-type φX174 RFI DNA. Phage DNA derived from single plaque lysates of the other four mutant complementary strand DNA preparations yielded exclusively homoduplex DNA after annealing with wild-type complementary strand DNA. It is concluded that priming with these deoxyhexadecamers resulted in the synthesis of complementary φX174 DNA with lethal mutations. The implications of these results, the construction of two silent, viable φX174 origin mutants and the failure to detect four others, for the initiation mechanism of φX174 RF DNA replication are discussed.

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.

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.

Islet amyloid polypeptide (IAPP) is synthesized in the islets of Langerhans

SummaryWe investigated the localization of IAPP mRNA by means of in situ hybridization in tissue sections of rat pancreas. A 35S-labeled, IAPP-specific DNA probe — hybridized specifically in the islets of Langerhans. This localization was confirmed by immunohistochemical localization of insulin and IAPP polypeptides on adjacent tissue sections. Moreover, combined in situ hybridization of IAPP mRNA and immunohistochemistry of insulin and IAPP polypeptide on the same section, using insulin as specific marker shows the presence of IAPP mRNA in the islets of Langerhans.

Sequence organization of a viral DNA insertion present in the adenovirus-type-5-transfonned hamster line BHK268-C31

The hamster cell line BHK268-C31 contains two large viral inserts which both include sequences from the left-hand end of adenovirus type 5 (Ad5) DNA. One of these viral inserts has been cloned in the lambda vector Charon 4A. Electron microscopic analysis and restriction enzyme mapping shows that the recombinant carries a 4.4-kb-long colinear segment of viral DNA, which is located between map positions 1.5 and 14.2 in the Ad5 genome. The junctions between viral DNA and flanking sequences have been sequenced and found not to show any specific features. One of the junctions is located in the E1a coding region, 573 bp from the left-hand end of the Ad5 genome, whereas the other junction is situated in the coding region for polypeptide IVa2. The promoter region as well as the cap site for the mRNAs from region E1a are thus missing from this insert and its role in viral transformation is unclear.

Alteration of the ATG start codon of the A* protein of bacteriophage ϕX174 into an ATT codon yields a viable phage indicating that A* protein is not essential for ϕX174 reproduction

Bacteriophage ϕX174 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 ϕX174, however, is unknown. Using oligonucleotide‐directed mutagenesis a viable ϕXl74 mutant was constructed in which the ATG start codon of the A* protein was changed into an ATT codon. This mutant, ϕX‐4499T, does not synthesize A* protein. The burst size of ϕX‐4499T amounted to 50% of that of wild type ϕXI74. This indicates that A* protein, although advantageous for phage reproduction, is not essential during the life cycle of bacteriophage ϕX174.

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.