Dana M. Fowlkes

An M13 phage library displaying random 38-amino-acid peptides as a source of novel sequences with affinity to selected targets

We have examined the potential of isolating novel ligands from a library of M13 pIII-fusion phage displaying peptides composed of 38 random amino acids (aa). The library was panned with streptavidin (SA) and a polyclonal goat antimouse IgG Fc antibody (Ab) preparation coupled to paramagnetic beads. SA selected two classes of phage from the library. One class exhibited the aa motif, HP(Q/M) theta (where theta signifies a non-polar aa), similar to the motif identified by Devlin et al. [Science 249 (1990) 404-406] using a 15-aa random peptide library displayed on phage. The other class of phage had no discernible motif. In binding experiments, the non-HP(Q/M) theta phage had a slightly higher affinity for SA than did the motif phage. Both classes of SA-binding phage failed to bind native and non-glycosylated forms of avidin, even though SA and avidin are structurally similar and both proteins possess extraordinary affinities for biotin. The polyclonal goat anti-mouse IgG Fc Ab preparation selected phage displaying sequences similar to a region of the mouse IgG Fc. Thus, a single immunodominant epitope on the mouse IgG Fc was identified. Furthermore, a second phage displaying peptides with no discernible sequence similarities to mouse IgG Fc was isolated. Thus, an M13 library displaying 38-aa peptides can yield phage with affinity for various targets. Finally, we have observed a biological bias against odd numbers of Cys residues in the displayed peptides.

Evolution and structure of the fibrinogen genes: Random insertion of introns or selective loss?

Chromosomal linkage as well as sequence homologies provide unequivocal evidence that the genes for the alpha, beta and gamma chains of fibrinogen arose by successive duplication of a single ancestral gene. Yet, when the three fibrinogen chains are aligned by amino acid homology, the positions of intervening sequences coincide at only two positions for all three chains. While one additional intron occurs at a homologous site in the beta and gamma chains, none of the positions of the remaining 11 introns in the three genes is shared. This arrangement of introns in the three fibrinogen genes suggests that either introns were selectively lost, implying that there is essential information in the retained introns, or the common introns were present in the ancestral fibrinogen gene and introns have been randomly inserted since the triplication of the original gene. The more likely possibility of selective loss of introns implies that the ancestral gene, as it existed about one billion years ago, must have been composed of numerous small exons.

The utility of a HindIII polymorphism of factor VIII examined by rapid DNA analysis

Summary. A previously described HindIII restriction fragment length polymorphism (RFLP) of factor VIII (FVIII) has its polymorphic site in the unsequenced nineteenth intron. We have located the polymorphic site, as well as an invariant site, by amplifying and sequencing IVS 19 using the polymerase chain reaction (PCR). The oligonucleotide primers were synthesized from known FVIII sequence on either side of the 19–20 splice junction. The amplified product was cloned into a plasmid and sequenced by the dideoxy chain termination method. The polymorphic HindIII site was 103 bp and the invariant site 184 bp from the 3’end of the nineteenth exon.

Analysis of centromere function in Saccharomyces cerevisiae using synthetic centromere mutants

We constructed Saccharomyces cerevisiae centromere DNA mutants by annealing and ligating synthetic oligonucleotides, a novel approach to centromere DNA mutagenesis that allowed us to change only one structural parameter at a time. Using this method, we confirmed that CDE I, II, and III alone are sufficient for centromere function and that A+T-rich sequences in CDE II play important roles in mitosis and meiosis. Analysis of mutants also showed that a bend in the centromere DNA could be important for proper mitotic and meiotic chromosome segregation. In addition we demonstrated that the wild-type orientation of the CDE III sequence, but not the CDE I sequence, is critical for wild-type mitotic segregation. Surprisingly, we found that one mutant centromere affected the segregation of plasmids and chromosomes differently. The implications of these results for centromere function and chromosome structure are discussed.

Construction and functional analysis of hybrid interleukin-6 variants Characterization of the role of the C-terminus for species specificity

We have constructed several hybrid human interleukin‐6 (IL‐6) variants in which the carboxyl‐terminus, which includes a receptor binding site of IL‐6 has been replaced with the C‐terminus of various proteins homologous to human IL‐6, IL‐6 hybrids with the C‐terminus of human growth hormone and human granulocyte‐colony stimulating factor maintain part of the biological activity of human IL‐6. Replacing the C‐terminus of human IL‐6 with the C‐terminus of mouse and rat IL‐6 resulted in a normal or increased activity on a mouse cell line; however, this gave a low (to 200‐fold less) activity on a human cell line compared to wild‐type human IL‐6. We therefore conclude that the C‐terminus of IL‐6 plays in important role in the species specificity of IL‐6.

Analysis of the heterogeneity of the biological responses to native and mutant human interleukin-6.

The structure‐function relationships of the biological activities of mutant varieties of the pleiotropic cytokine interleukin‐6 (human) were measured by three assays: induction of immunoglobulin M (IgM) secretion from an Epstein‐Barr virus‐transformed human B cell line and induction of fibrinogen secretion from either a human hepatoma cell line or a rat hepatoma cell line. The biological effects of the cytokine were characterized by three parameters as determined by a novel analysis: effectiveness (the maximal response attainable), efficiency (the concentration yielding a half‐maximal response), and complexity (a measure of heterogeneity and feedback control). Substitution of serine for cysteine was associated with a reduction in the effectiveness of interleukin‐6 in both fibrinogen secretion assays. In the assay with human hepatoma cells, there was also a profound reduction in efficiency. Serine substitution in the human IgM synthesis assay appears mainly to reduce the efficiency. Deletion of amino acids 4 to 23 increased the efficiency in the rat hepatoma assay. The complexity parameter suggests the presence of multiple receptor classes or negative feedback in all three assays. Use of the proposed sequential approach to the analysis of dose‐response relations in bio‐ assays provides a more useful quantitative assessment of activities as well as more insight into the complexity of the reactions.