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Accurate and quantitative transcription of the tk gene requires sequence elements that reside within 110 nucleotides of 5'-flanking DNA. We have determined the boundaries of a hypersensitive region in 5'DNA flanking the tk gene by analyzing the relative sensitivity of specific restriction sites clustered in this region. Five different restriction enzymes, recognizing over 50 sites in the promoter region of the tk gene, each show a preferential cleavage in nuclei at a restricted number of sites residing between positions -4 and -182. Thus the tk promoter sequences are contained entirely within a hypersensitive region. Analysis of this site in tk+ transformants, a tk- mutant and tk+ rerevertant indicates that expression of tk RNA is correlated with structural alterations in the tk promoter.

The kappa immunoglobulin (Ig) genes from rat kidney and from rat myeloma cells were cloned and analyzed. In kidney DNA one C kappa species is observed by Southern blotting and cloning in phage vectors; this gene most likely represents the embryonic configuration. In the IR52 myeloma DNA two C kappa species are observed: one in the same configuration seen in kidney and one which has undergone a rearrangement. This somatic rearrangement has brought the expressed V region to within 2.7 kb 5' of the C kappa coding region; the rearrangement site is within the J kappa cluster which we have mapped. The rat somatic Ig rearrangement, therefore, closely resembles that seen in mouse Ig genes. In the rat embryonic fragment two J kappa segments were mapped at 2 and 4.3 kb 5' from the C kappa coding region. Therefore, the rat J kappa cluster extends over about 2.3 kb, a region much longer than the 1.4 kb of the mouse and human J kappa clusters. In the region between C kappa and the expressed J kappa of IR52 myeloma DNA, and XbaI site present in the embryonic kappa gene has been lost. A somatic mutation has therefore occurred in the intervening sequence DNA approx. 0.7 kb 3' from the V/J recombination site. Southern blots of rat kidney DNA hybridized with different rat V kappa probes showed non-overlapping sets of bands which correspond to different subgroups, each composed of 8-10 closely related V kappa genes.

Transformation, or DNA-mediated gene transfer, permits the stable introduction of new genetic information into a cell and therefore provides an opportunity to examine the expression of exogenous DNA sequences in the transformed host. A series of deletion mutants to analyze the sequences required for accurate and quantitative in vivo transcription of the herpes simplex virus (HSV) thymidine kinase (tk) gene has been constructed. Control of the efficiency of transcription as well as rough specification of the sites of initiation reside within sequences 40-110 nucleotides from the tk structural gene. The sequences responsible for induction of tk mRNA overlap this transcriptional control region. Analysis of the conformation of the integrated tk gene in the chromosome reveals that these DNA sequences are exquisitely sensitive to nuclease attack. The major heat shock gene of Drosophila and the human beta globin gene have also been introduced into murine cells to identify sequences responsible for induction of these finely regulated genes.

Sequences in the human genome with homology to the murine mammary tumor virus (MMTV) pol gene were isolated from a human phage library. Ten clones with extensive pol homology were shown to define five separate loci. These loci share common sequences immediately adjacent to the pol-like segments and, in addition, contain a related repeat element which bounds this region. This organization is suggestive of a proviral structure. We estimate that the human genome contains 30 to 40 copies of these pol-related sequences. The pol region of one of the cloned segments (HM16) and the complete MMTV pol gene were sequenced and compared. The nucleotide homology between these pol sequences is 52% and is concentrated in the terminal regions. The MMTV pol gene contains a single long open reading frame encoding 899 amino acids and is demarcated from the partially overlapping putative gag gene by termination codons and a shift in translational reading frame. The pol sequence of HM16 is multiply terminated but does contain open reading frames which encode 370, 105, and 112 amino acid residues in separate reading frames. We deduced a composite pol protein sequence for HM16 by aligning it to the MMTV pol gene and then compared these sequences with other retroviral pol protein sequences. Conserved sequences occur in both the amino and carboxyl regions which lie within the polymerase and endonuclease domains of pol, respectively.

Sequence determination of the joining segment gene (J) cluster in the kappa chain (J kappa) in the embryonic context demonstrates that rat genome contains seven J kappa gene segments that expanded from an ancestral cluster of five J kappa genes. The rat J segments are separated by about 300 base pairs (bp) and are flanked 5' by the presumed variable region (V)/J recombination signal sequence and 3' by the RNA splicing signal. Two of the J gene segments designated J2A and J2B and their 5'-flanking spacer DNA bear striking homology to J2 and its 5'-flanking spacer. Thus, the unit of duplication was the entire J kappa coding region and 5' noncoding spacer (345 bp). The duplication probably occurred as two separate unequal crossing-over (UXO) events. The first UXO event can be confined to recombination within an identical stretch (14 bp long) located at the 3' ends of the coding regions of J1 and J2. The second event could involve a longer segment (372 bp) of tight homology generated by the first UXO event, thus increasing the probability of repeated expansion of the same DNA segment. The sequence homology among the rat duplicated segments (98-99%) is larger than the homology between the corresponding rat and mouse segments (89%), showing that the rat J kappa gene expansion must have occurred after rat and mouse divergence 10 X 10(6) yr ago. We estimate that the first and second UXO events occurred 2 X 10(6) and 1 X 10(6) yr ago, respectively. J3 of rat and mouse share the same mutation (G leads to C) in the RNA splicing signal that presumably inactivates J3. This mutation preceded divergence of the two species. A mutation in the first nucleotide of codon 96 has occurred in both duplicated segments, the only position along 345 bp where J2, J2A, and J2B differ from each other. This results in three different amino acids at position 96 not present in any other J kappa. These mutations are physiologically significant because they diversify the third complementarity-determining region (CDR3) and, thus, may reflect selective pressure to increase antibody diversity. The germ-line diversification of CDR3 was exercised within the last 1-2 X 10(6) yr.

Transgenic mice were generated carrying either the long terminal repeat of Human Immunodeficiency Virus fused to the bacterial chloramphenicol acetyl transferase reporter gene or a control element of the murine alpha A crystallin gene fused to the tat gene of human immunodeficiency virus. By crossing these two strains, progeny were obtained which carried both transgenes. The bacterial reporter gene was specifically transactivated in the eyes of these animals.