Making antibody fragments using phage display libraries

To by-pass hybridoma technology and animal immunization, we are trying to build antibodies in bacteria by mimicking features of immune selection. Recently we used fd phage to display antibody fragments fused to a minor coat protein, allowing enrichment of phage with antigen. Using a random combinatorial library of the rearranged heavy (VH) and kappa (V kappa) light chains from mice immune to the hapten 2-phenyloxazol-5-one (phOx), we have now displayed diverse libraries of antibody fragments on the surface of fd phage. After a single pass over a hapten affinity column, fd phage with a range of phOx binding activities were detected, at least one with high affinity (dissociation constant, Kd = 10(-8) M). A second pass enriched for the strong binders at the expense of the weak. The binders were encoded by V genes similar to those found in anti-phOx hybridomas but in promiscuous combinations (where the same V gene is found with several different partners). By combining a promiscuous VH or V kappa gene with diverse repertoires of partners to create hierarchical libraries, we elicited many more pairings with strong binding activities. Phage display offers new ways of making antibodies from V-gene libraries, altering V-domain pairings and selecting for antibodies with good affinities.     

Glyoxalase II activity in tumours

Résumé Les auteurs ont constaté que l'activité de la glyoxalase II, très intense dans les tissues normaux, est au contraire très faible dans les cellules et tissus cancéreux, où elle peut même manquer complètement.

---

---

This work was supported by VI Department Medical Sciences of the Polish Academy of Science.     

Spontaneous functional correction of homozygous fanconi anaemia alleles reveals novel mechanistic basis for reverse mosaicism.

Somatic mosaicism due to reversion of a pathogenic allele to wild type has been described in several autosomal recessive disorders. The best known mechanism involves intragenic mitotic recombination or gene conversion in compound heterozygous patients, whereby one allele serves to restore the wild-type sequence in the other. Here we document for the first time functional correction of a pathogenic microdeletion, microinsertion and missense mutation in homozygous Fanconi anaemia (FA) patients resulting from compensatory secondary sequence alterations in cis. The frameshift mutation 1615delG in FANCA was compensated by two additional single base-pair deletions (1637delA and 1641delT); another FANCA frameshift mutation, 3559insG, was compensated by 3580insCGCTG; and a missense mutation in FANCC(1749T-->G, Leu496Arg) was altered by 1748C-->T, creating a cysteine codon. Although in all three cases the predicted proteins were different from wild type, their cDNAs complemented the characteristic hypersensitivity of FA cells to crosslinking agents, thus establishing a functional correction to wild type.     

X-linked inheritance of Fanconi anemia complementation group B

Fanconi anemia is an autosomal recessive syndrome characterized by diverse clinical symptoms, hypersensitivity to DNA crosslinking agents, chromosomal instability and susceptibility to cancer. Fanconi anemia has at least 11 complementation groups (A, B, C, D1, D2, E, F, G, I, J, L); the genes mutated in 8 of these have been identified. The gene BRCA2 was suggested to underlie complementation group B, but the evidence is inconclusive. Here we show that the protein defective in individuals with Fanconi anemia belonging to complementation group B is an essential component of the nuclear protein 'core complex' responsible for monoubiquitination of FANCD2, a key event in the DNA-damage response pathway associated with Fanconi anemia and BRCA. Unexpectedly, the gene encoding this protein, FANCB, is localized at Xp22.31 and subject to X-chromosome inactivation. X-linked inheritance has important consequences for genetic counseling of families with Fanconi anemia belonging to complementation group B. Its presence as a single active copy and essentiality for a functional Fanconi anemia-BRCA pathway make FANCB a potentially vulnerable component of the cellular machinery that maintains genomic integrity.     

Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome

In fruit fly research, chromosomal deletions are indispensable tools for mapping mutations, characterizing alleles and identifying interacting loci. Most widely used deletions were generated by irradiation or chemical mutagenesis. These methods are labor-intensive, generate random breakpoints and result in unwanted secondary mutations that can confound phenotypic analyses. Most of the existing deletions are large, have molecularly undefined endpoints and are maintained in genetically complex stocks. Furthermore, the existence of haplolethal or haplosterile loci makes the recovery of deletions of certain regions exceedingly difficult by traditional methods, resulting in gaps in coverage. Here we describe two methods that address these problems by providing for the systematic isolation of targeted deletions in the D. melanogaster genome. The first strategy used a P element-based technique to generate deletions that closely flank haploinsufficient genes and minimize undeleted regions. This deletion set has increased overall genomic coverage by 5-7%. The second strategy used FLP recombinase and the large array of FRT-bearing insertions described in the accompanying paper to generate 519 isogenic deletions with molecularly defined endpoints. This second deletion collection provides 56% genome coverage so far. The latter methodology enables the generation of small custom deletions with predictable endpoints throughout the genome and should make their isolation a simple and routine task.     

Removal of bound calcium from nematocyst contents causes discharge

Nematocysts are the stinging organelles of jellyfish, sea anemones and other cnidarians. Each one consists of a closed capsule filled with fluid. In the resting state, part of the surface of the capsule is inverted, forming a tubular thread, which is everted explosively on excitation. The mechanism of explosion is not yet understood, it may be relevant to exocytosis in general, as the nematocyst is a specialized type of exocytotic secretion originating in the Golgi apparatus. Picken and Skaer observed that the capsular fluid showed a large depression of freezing point, suggesting that the osmotic pressure might be as high as 140 bar. They tentatively ascribed the explosion to a sudden increase in permeability of the capsule wall, allowing a rapid osmotic influx of water. However, there is evidence that the material of the thread itself may be capable of some degree of extension and it has been suggested that osmosis plays no part in discharge. We present here a new theory: that the capsule wall is permeable to water even in the undischarged state; discharge is initiated by an increase in the osmotic pressure of the capsular fluid which is brought about by removal of bound calcium ions.     

Lag period of action of 25-hydroxycholecalciferol on bone collagen metabolism in vitamin D deficient rats

Zusammenfassung Nachweis, dass die Freisetzung von Hydroxyprolin bei Knochenfragmenten bereits 6 h nach i.v. Verabreichung von 2,5 g 25-Hydroxycalciferol signifikant erhöht war, während eine entsprechend zugeführte Dosis von Vitamin D₃ erst 13 h später deutlich wirkte.

---

---

We thank Dr.E. Kodicek and Dr.E. M. Cruickshank of Dunn Nutritional Laboratory for help and criticism, and Mrs.J. Jobbágy for technical assistance.