No abstract

A competition hybridization strategy using size-cut cDNA libraries as both probe and competitor was designed for the cloning of genes whose mRNAs are either regulated transcriptionally or vary in abundance as a function of cell line or cell cycle. We used this strategy to construct cDNA libraries from a particular size fraction of mRNA from three members of a xeroderma pigmentosum (XP), complementation group A family. Size-cut cDNA libraries derived from the father's, mother's, and child's fibroblast cell line were used in a competition scheme to screen two lambda gt11 human cDNA libraries. Of the 15 positive lambda gt11 clones which have been characterized, at least 14 clones represent the same gene which is present in greater abundance in both the mother and father XP obligate heterozygotes relative to the homozygous affected child.

Both the male and the female of Mastomys natalensis, an African rat, have high levels of nerve growth factor (NGF) in their submaxillary glands. Mastomys high molecular weight (HMW) NGF was purified by gel filtration and ion-exchange chromatography and was compared with HMW NGF from the male mouse submaxillary gland. Mastomys HMW NGF sediments as a 5S species, does not exhibit esterase activity, and is more difficult to dissociate at acid pH than mouse 7S NGF. The biological activity could be isolated as a purified Mastomys beta NGF protein identical in size and charge with that purified from male mice. The N-terminal amino acid sequence of the first 20 residues was determined and found to differ from that of mouse only at residue 8. Western blotting of Mastomys 5S NGF using antiserum against mouse beta NGF indicates that the beta NGF subunit of Mastomys is very similar to that of the mouse. Southern blots using a mouse kallikrein probe also demonstrate the presence of a large kallikrein family in Mastomys similar to that in mouse, and Northern blots verify transcription of kallikreins in Mastomys submaxillary gland. SDS-PAGE and isoelectric focusing gels reveal a Mastomys subunit that comigrates with mouse alpha subunit. However, neither oligonucleotide probes directed against mouse alpha subunit RNA nor antibodies directed against mouse alpha NGF cross-react strongly with the Mastomys material. This indicates that the second subunit of the Mastomys complex is not very similar to the mouse alpha subunit.

A recent report (1) presented evidence for allosterism in reverse transcription by Mason-Pfizer monkey virus reverse transcriptase and by E. coli DNA polymerase I. Our experiments also demonstrate these apparent cooperative effects when synthesis is catalyzed by either avian myeloblastosis virus DNA polymerase, feline sarcoma virus DNA polymerase, or E. zoli DNA polymerase I (large fragment). We show that the apparent cooperativity depends on the use of oligo(dT)12-18 as primer. However, if the polymerase reaction products are isolated chromatographically, then the polymerases obey classical MichaelisMenten kinetics with respect to substrate and enzyme concentrations. These results suggest that the cooperative effects are an acid precipitation artifact. The results are also consistent with the enzyme operating by a distributive mechanism with the oligo(dT)12-18 primer.