Immunologic memory to phosphocholine keyhole limpet hemocyanin. Recurrent mutations in the lambda 1 light chain increase affinity for antigen.

Anti-phosphocholine (PC)-keyhole limpet hemacyanin hybridomas representative of a memory response that express the lambda 1 L chain isotype have a high reactivity to PC-protein. A common feature of these hybridomas possessing high affinity for PC-protein is the occurrence of somatic mutations resulting in replacement changes in three CDR2 positions of the lambda 1 L chain. The influence of each of these three positions on the Ag binding properties of these antibodies was examined by site-specific mutagenesis and expression of recombinant antibody molecules by transfected cells. Affinity measurements and fine specificity profile determinations demonstrated the importance of the three lambda 1 CDR2 positions in Ag binding. Compared to antibodies expressing germline lambda 1, including one with an additional junctional serine that is not encoded by V or J, those antibodies possessing critical changes in CDR2 would have a strong selective advantage based on affinity differences for Ag. Sequence analysis of a group of clonally related hybridomas expressing mutated lambda 1 genes allowed construction of a hypothetical genealogic tree that suggests selection based on changes in CDR2 of lambda 1 in the absence of H chain mutations. The results are consistent with stepwise acquisition of mutations and selection based on affinity constraints.

The SV40 nucleosome-free region is detected throughout the virus life cycle.

The structures of SV40 intracellular chromatin complexes and of extracellular virus particles were examined by photolabeling with a radioactive psoralen derivative in order to determine the fate of the exposed origin region during the virus life cycle. We have previously shown that the origin region of intracellular SV40 chromatin is preferentially accessible to psoralen derivatives in vivo, whereas psoralen adducts are uniformly distributed when purified virus particles are photoreacted. We demonstrate here that when virion is photoreacted prior to a freeze-thaw cycle, the exposed regulatory region detected in intracellular nucleoprotein complexes is also found in mature virus particles. In contrast, if the virion is frozen and thawed prior to the photoreaction, the origin is not preferentially exposed to photoaddition. Virus particles that have not been subjected to a freeze-thaw cycle were found to exhibit preferential labeling in the origin region whether they were irradiated intracellularly, in culture medium, or following purification. Banding the virus in CsCl had no significant effect on the relative accessibility of the origin region to psorealen. Our findings indicate that the open regulatory region found on intracellular SV40 chromatin persists throughout the virus life cycle.

A mapped set of DNA markers for human chromosome 17.

We have developed and mapped by genetic linkage a primary set of markers for chromosome 17. The map consists of 21 loci derived from 27 probe/enzyme systems, including eight highly informative markers at loci containing a variable number of tandemly repeated DNA sequences (VNTRs). The map is continuous from the telomeric region of the short arm to the telomeric region of the long arm, covering estimated genetic distances of 218 cM in males and 279 cM in females. The average heterozygosity among all 21 loci in the population sample analyzed is 58%; 77% heterozygosity was observed among the eight VNTR markers that were highly informative. This map will make it possible to detect by linkage the location of genetic defects associated with chromosome 17 and will also provide anchor points for a high-resolution map of this chromosome.

Chromosomal localization of the human proenkephalin and prodynorphin genes.

DNA probes derived from rat and human proenkephalin and prodynorphin genes have been used to localize these two opiate neuropeptide genes on human chromosomes. Hybridization of probes to Southern blots made with DNAs from a rodent-human somatic-cell hybrid panel indicates localization of proenkephalin to human chromosome 8 and of prodynorphin to human chromosome 20. In situ hybridization to metaphase chromosomes confirms these assignments and indicates regional localizations of proenkephalin to 8q23-q24 and of prodynorphin to 20p12-pter. A human genomic prodynorphin clone reveals a frequent two-allele TaqI polymorphism.

SV40 virus particles lack a psoralen-accessible origin and contain an altered nucleoprotein structure.

The nucleoprotein structure of SV40 virions was examined by photolabeling purified virus with the radioactive psoralen derivative hydroxymethyltrimethylpsoralen (HMT). Unlike SV40 chromatin in situ, the viral origin region is not preferentially accessible to drug addition. The ratio of the distribution of radioactivity in the DNA restriction fragments of virion DNA to that of purified SV40 DNA demonstrates that the photoadducts are positioned similarly on the circular molecule in both samples. Virion purified from infected cells was also analyzed for the presence of an open region and found to exhibit the same pattern of [3H]HMT addition as mature extracellular virion. The nucleosome-free region detected at the SV40 replication origin in intracellular minichromosomes is not present in either population of intact virus particles. We also examined the level of drug addition obtained when purified virion or SV40-infected cells were treated with saturating doses of [3H]HMT. Marked differences in the plateau levels of bound drug indicate that an altered nucleoprotein structure exists in SV40 virions that does not protect the DNA from photoaddition to the same extent as do the nucleosomes of intracellular SV40 DNA.

Studies of a synthetic substrate in canine globoid cell leukodystrophy.

Gal et al. ((1977) Clin. Chim. Acta 77, 53-59) reported the use of a new synthetic substrate, 2-hexadecanoylamino-4-nitrophenyl-beta-D-galactopyranoside for the diagnosis of human globoid cell leukodystrophy. Assay of beta-galactosidase in brain homogenates from normal, carrier, and globoid cell leukodystrophy-affected dogs utilizing this new substrate demonstrated overlapping activities. Instead of reflecting specific D-galactosyl-N-acylsphingosine galactohydrolase (EC 3.2.1.46), the 2-hexadecanoylamino-4-nitrophenyl-beta-D-galactopyranoside beta-galactosidase activity in canine brain is highly correlated with nonspecific 4-methylumbelliferyl beta-galactosidase. Optimization of the 2-hexadecanoyl-amino-4-nitrophenyl-beta-D-galactopyranoside assay system for canine brain and the use of varying concentrations of taurocholate or taurodeoxycholate in the assay mixture did not alter the lack of specificity. These results indicate a significant difference in the nature of the underlying defect in galactosylceramide beta-galactosidase in canine globoid cell leukodystrophy compared to human globoid cell leukodystrophy.

Replication of mammalian DNA in bromodeoxyuridine: appearance of a component with intermediate density.

When three lines of mammalian cells were cultured with 5-bromodeoxyuridine (BrdUrd) for less than one generation, their DNAs displayed three peaks in CsCl gradients. In addition to the expected unsubstituted (LL) and hybrid (LH) peaks, there was a significant absorbance peak of intermediate density (INT) between LH and LL DNAs. This INT DNA has characteristics expected of an intermediate of DNA replication. Upon shearing, it behaves as though it contains contiguous segments of unsubstituted and hybrid DNAs. Upon continuous exposure of cells to [3H]-BrdUrd, radioactivity accumulates in INT DNA for 60-90 min when a steady state condition is reached. At that time, the rate of incorporation into LH DNA increases, consistent with a precursor-product relationship. In a pulse-chase experiment, radioactivity is chased from INT DNA into LH DNA. To account for the above observations and for the size and sharpness of the INT DNA peak in CsCl, we suggest that a high molecular weight replication intermediate accumulates before completing replication into mature daughter molecules.