Bjørn H. Lindqvist

A region from the medium chain adaptor subunit (mu) recognizes leucine- and tyrosine-based sorting signals.

Tyrosine-based sorting signals in the cytosolic tails of membrane proteins have been found to bind directly to the medium chain subunit (μ) of the adaptor complexes AP-1 and AP-2. For the leucine-based signals, an interaction with AP-1 and AP-2 has been reported, but no specific interacting subunit has been demonstrated. After searching for molecules interacting with the leucine-based sorting signals within the cytosolic tail of the major histocompatibility complex class II-associated invariant chain using a phage display approach, we identified phage clones with homology to a conserved region of the AP-1 and AP-2 μ chains. To investigate the relevance of these findings, we have expressed regions of mouse μ1 and μ2 chains on phage gene product III and investigated the binding to tail sequences from various transmembrane proteins with known endosomal targeting signals. Enzyme-linked immunosorbent binding assays showed that these phages specifically recognized peptides containing functional leucine- and tyrosine-based sorting signals, suggesting that these regions of the μ1 and μ2 chains interact with both types of sorting signals.

Bacteriophage P2 and P4 morphogenesis : protein processing and capsid size determination

An interesting feature of the bacteriophage P2-P4 system is the switch in size between a large P2 (60 nm) and a small P4 (45 nm) capsid. We have investigated whether the protein processing reactions cleaving the primary translation product gpN to several capsid proteins (h1, h2, and N*) are involved in this switch. Using antibodies specific against gpN and its derivatives we have identified all the structural components of mature P2 and P4 particles that are derived from gpN. Our estimate of the relative amounts of gpN derivatives suggests that the previously identified minor capsid proteins h1 and h2 can only be essential structural components of the P4, and not the P2, capsid. Nevertheless, the relative amounts are similar in vivo during a P2 and a P4 infection. This indicates that the switch in head size is not caused by the presence of elevated amounts of h1 and h2 during P4 morphogenesis. We have also identified the sites where gpN is cleaved to its derivatives h1, h2, and N*, ascertaining that the cleavage sites are the same in P2 and P4. Our results indicate that the processing reactions are not directly involved in the head size determination mechanism.

Identification and Characterisation of C1q-Binding Phage Displayed Peptides

Five phage displayed peptide libraries were screened for binders to C1q, the recognition subunit of the classical complement pathway. Two rounds of panning resulted in the isolation and characterisation of several different phage displayed C1q-binding peptides from all five libraries. Two groups of the characterised peptides show sequence similarity with part of the metal ion dependent adhesion site (MIDAS) of integrin A-domains, and the site 187LRNPCPNKEKECQPPF of CD18 (integrin beta2), respectively. These results support binding of complement receptor 3 (CR3, CD11b/CD18, Mac1) to C1q and further suggest C1q binding sites in CR3. We also discuss sequence matches between the characterised peptides and proteins known to interact with C1q, as well as other proteins listed in the SwissProt databank. These findings are of interest for the study of the complement system and may lead to the development of peptides, fusion products or peptido-mimetics with C1q modulating potential.

Asymmetric transcription of the coliphage P2 genome during infection

Abstract Transcription of the coliphage P2 genome during infection has been studied by RNA-DNA hybridization, employing DNA strands separated in the presence of poly-(UG). The results indicate that P2 transcription is highly asymmetric, i.e., about 95% of the P2 mRNA synthesis occur from the “heavy” strand and only 5% from the “light” strand. This strand distribution of P2 mRNA synthesis appears to be constand during the entire infectious process. Measurements of P2 transcription in the absence of P2 DNA replication, i.e., under nonpermissive conditions with two early mutants (DNA defective) and during infection of a REP − host with P2, indicate that the rate of the initial transcription is normal. The rate of transcription late in infection, however, is greatly reduced compared to the normal P2 infection. The strand distribution of P2 mRNA synthesis under these conditions is the same as that observed in the wild-type P2 infection. Satellite phage P4 activated transcription from P2 prophage has been detected. The rate of this transcription may not be widely different from that observed during lytic P2 infection.

Detection of genital papillomavirus types by polymerase chain reaction using common primers

We describe the detection of eight genital human papillomavirus (HPV) types, including HPV16 and HPV18, by PCR amplification of a 323 base‐pair region of the genome within the LI open reading frame (ORF). The primer sequences are: TGYAAATATCCWGATTWTWT and GTATCWACMA‐CAGTAACAAA. The method will detect purified HPV16 DNA down to a concentration of as little as a single molecule in 100 μl. The method is also applicable to purified DNA and crude lysates from tumour biopsies. Typing of the PCR product can be achieved with specific oligonucleotide probes.

Human papillomavirus infection in Norwegian women with cervical cancer

The objective of the present study was to determine the prevalence of human papillomavirus (HPV) infections in Norwegian women with cervical cancer. We used the polymerase chain reaction (PCR) and Southern blot techniques to assess the prevalence of HPV in cervical biopsies of 133 women admitted to the Norwegian Radium Hospital for treatment of cervical cancer. At the time of sampling (from February 1988 to April 1989) about 85% of Norwegian women with cervical cancer were treated at the Norwegian Radium Hospital. HPV was found in biopsies of 91 (68%) of women with cancer; 70 (53%) biopsies contained HPV type 16, 19 (14%) HPV type 18, 4 (3%) HPV type 33, 2 (1.5%) HPV type 11, and 3 (2%) HPV DNA of unknown type (HPVX). Five percent of biopsies were doubly infected, chiefly with HPV 16 + 18. We found a significant association between HPV 18 and low age, poorly differentiated tumors and adenocarcinomas. Our results show that there is an association between HPV types 16 and 18 and cervical cancer also in a Norwegian setting. PCR was more sensitive than Southern blotting for detection of HPV. Thirty‐six (27.5%) of cancer biopsies were positive by PCR but negative by Southern blotting, as against 49 (73.5%) positive by both methods; we also encountered 4 samples positive by Southern blotting and negative by PCR. In 23/53 cancer biopsies positive by Southern blotting we found evidence for integrated or rearranged HPV genomes.

Selection of phage displayed peptides from a random 10-mer library recognising a peptide target

BACKGROUND Peptide display libraries are powerful tools in the search for detailed information about protein-protein interactions. Usual targets for isolation of phage displayed peptide ligands include antibodies, various receptors, other full size proteins or larger fragments thereof. Smaller protein fragments such as synthetic peptides have not been reported as targets for screening of peptide display libraries. OBJECTIVES To investigate whether a protein target used for screening of a peptide display library could be scaled down to peptide size. As the peptide target we wanted to use a sequence derived from the cytosolic tail of MHC class II associated invariant chain containing a leucine class endosomal sorting signal, known to be recognised as an autonomous functional unit during targeting of class II complexes to antigen processing compartments. STUDY DESIGN A screening procedure where a synthetic 15-mer invariant chain peptide was coupled to a methacrylate matrix of high binding capacity was developed, and three rounds of selection were performed from a random 10-mer fUSE5 display library. RESULTS The peptide display library was successfully enriched for phage clones with affinity for the invariant chain peptide. Furthermore, the binding phage clones were able to distinguish between a functional and a mutated form of the target. These clones therefore displayed possible peptide mimetics of signal recognition sites in the cellular sorting machinery. CONCLUSION The size of a protein target may be scaled down to peptide size and be recognised by a 10-mer peptide displayed on filamentous phage. This approach may particularly be useful when the peptide target contains a functional unit for recognition.

The polarity suppression factor of bacteriophage P4 is also a decoration protein of the P4 capsid.

We show that the product of the polarity suppression (psu) gene from bacteriophage P4 associates with P4 capsids. This association can occur when Psu is (i) provided in vivo from the P4 genome or from a plasmid or (ii) provided in vitro by mixing viable phage particles with Psu protein. Psu is unable to associate with the larger capsid of P4s helper phage P2. Discrimination of the P4 and P2 capsids by Psu appears to be independent of the presence of the P4 genome in the capsid, since P2 size capsids filled with P4 DNA cannot accommodate Psu association. P4 psu particles devoid of Psu are less stable than P4 particles carrying Psu. These results indicate that, in addition to its antitermination activity at Rho-dependent terminators, Psu is also a decoration protein that stabilizes the P4 capsids.

Bacteriophage and gene transfer

Viruses Viruses reproduce in a host cell. Hence, avirus can be considered a genetic package in pursuit of a cell to multiply in. This mode of multiplication make viruses natural agents of horizontal genome distribution. During the infection process the virus genomes will have a chance to recombine with the host genetic material including resident viruses, cryptic elements, transposable elements etc. resulting in genetic exchanges and rearrangements of the virus genome. Such genetic exchanges are the basis for virus mediated horizontal gene transfer or transduction. Plant and animal viruses are the obvious agents of gene transfer between plants and animals, respectively.