Correlation of gene and protein structure of rat and human lipocortin I.

Lipocortins (annexins) are a family of calcium-dependent phospholipid-binding proteins with phospholipase A2 inhibitory activity. The characteristic primary structure of members of this family consists of a core structure of four or eight repeated domains, which have been implicated in calcium-dependent phospholipid binding. In two lipocortins (I and II) a short amino-terminal sequence distinct from the core structure has potential regulatory functions which are dependent on its phosphorylation state. We have isolated the rat and the human lipocortin I genes and found that they both consist of 13 exons with a striking conservation of their exon-intron structure and their promoter and amino acid sequences. Both lipocortin I genes are at least 19 kbp in length with exons ranging from 57 to 123 bp interrupted by introns as large as 5 kbp. Each of the four repeat units of lipocortin I are encoded by two consecutive exons while individual exons code for the highly conserved putative calcium-binding domains. The promoter sequences in the rat and in human genes are highly conserved and contain nucleotide sequences characterized as enhancer sequences in other genes. The structure of the lipocortin I gene lends support to the hypothesis that the lipocortin genes arose by a duplication of a single domain.

The 0 degree C closed complexes between Escherichia coli RNA polymerase and two promoters, T7-A3 and lacUV5.

The promoter-specific binding of Escherichia coli RNA polymerase to the T7-A3 and the lacUV5 promoters at 0 degrees C was analyzed by DNase I footprinting. At 37 degrees C, the footprint from RNA polymerase bound to the A3 promoter is essentially the same as that reported by Galas, D.J., and Schmitz, A., (1978) Nucleic Acids Res. 5, 3157-3170 for the lacUV5 promoter. At 0 degrees C, the footprint for the A3 promoter is well defined but reduced in size. The principal difference between the 0 and 37 degrees C footprints is a region from -2 to +18 which is protected by polymerase at the higher but not at the lower temperature. In contrast, the 0 degree C footprint for the lacUV5 promoter differs substantially in character from the footprint for A3 at 0 degree C. The footprint is similar to the pattern of DNase I digestion of DNA bound to a surface; alternating regions of sensitive and protected DNA are spaced at intervals of about 10 base pairs. This region of DNase I-sensitive and -resistant DNA has the same boundaries as the 0 degree C footprint on T7-A3. Temperature shift experiments confirmed the sequence specificity of the RNA polymerase interaction with UV5 at 0 degree C. These results indicate that RNA polymerase binds specifically to each promoter sequence in a closed complex. The increased time and amounts of RNA polymerase required to form the 0 degree C footprint on the lacUV5 promoter indicate that it binds RNA polymerase more weakly than does the T7-A3 promoter. Therefore there is a correlation between the binding constant for closed complex formation estimated from kinetic measurements and the formation of the 0 degree C footprint. The -35 region of the promoter may be more important in establishing the 0 degree C footprint because the T7-A3 promoter is a better match to the consensus sequence. Conversely, the -10 region seems less important because lacUV5 is a perfect match to the consensus, whereas the T7-A3 promoter matches at only five out of seven positions. The 0 degree C footprints encompass both regions along with the spacer; the combination of these regions rather than an individual region may determine the character of the footprint and the magnitude of the binding constant.

Production of immunologically active surface antigens of hepatitis B virus by Escherichia coli.

Several plasmids have been constructed which direct the synthesis of hepatitis B virus surface antigens in Escherichia coli either as the native polypeptide or fused to other plasmid encoded polypeptides. When injected into rabbits, extracts from bacteria carrying some of these plasmids induced the synthesis of antibodies to the antigens even though the extracts did not give satisfactory positive results in radioimmunoassay for them. Either the NH2-terminal segment or the COOH-terminal segment of the surface antigens alone was sufficient to elicit the immune response, but antibodies against the two segments showed different specificities. The results emphasize the value of an in vivo assay for the presence of antigens in crude cell extracts and illustrate the feasibility of this type of screening with laboratory animals.

Sedimentation of homogeneous double-strand DNA molecules.

Sedimentation velocity studies have been carried out with isolated double-strand DNA fragments prepared by digestion of PM2 phage with the restriction endonuclease Hae III. The results show that DNA molecules shorter than about 200 base pairs behave almost exactly as rigid rods with a diameter of 27 A. The behavior of the larger fragments (up to 1735 base pairs) can be described very well by either the theory of Yamakawa and Fujii (Yamakawa, H., and Fujii, M. (1973), Macromolecules 6. 407) using the same diameter and a persistence length of 575 A, or the theory of Hearst and Stockmayer (Hearst, J.E., and Stockmayer, W.H. (1962), J. Chem. Phys. 37, 1425) using a diameter of 20 A and a persistence length of 525 A.

Quantitative analysis of the digestion of yeast chromatin by staphylococcal nuclease.

The DNA in intranuclear yeast chromatin is protected from rapid staphylococcal nuclease degradation so as to yield an oligomeric series of DNA sizes. The course of production and disappearance of the various oligomers agrees quantitatively with a theory of random cleavage by the enzyme at uniformly susceptible sites. The sizes of the oligomers are integral repeats of a basic size, about 160 base pairs, and 80-90% of the yeast genome is involved in this repeating structure. Within this repeat there exists a 140 base pair core of more nuclease-resistant DNA. During the course of digestion, the sizes of the oligomers decrease continuously. The widths of the distribution of DNA sizes increase in order: monomer (1 X repeat size, half width = 5-7 base pairs) less than dimer (2 X repeat size, half width = 30 base pairs) less than trimer (3 X repeat size, half width = 40-45 base pairs). The yeast genome thus seems to have variable spacing of the nucleaseresistant cores, to produce the average repeat size of about 160 base pairs. Also, the presence of more than one species of monomer and dimer at certain times of digestion suggests a possible heterogeneity in the subunit structure.

Comparative subunit structure of HeLa, yeast, and chicken erythrocyte chromatin.

We have compared the chromatin subunit structure of yeast, HeLa, and chicken erythrocyte by analyzing the DNA fragments produced by in situ digestion with staphylococcal nuclease (EC 3.1.4.7) and DNase I(EC 3.1.4.5). The repeat size of the chromatin varies among (and within two of) the three organisms but the size and the structure of the most nuclease-resistant "core" of the repeat is the same. Thus, the interspecies differences in repeat size are due to different lengths of nuclease-sensitive "spacer" DNA between the cores. There also seems to be a difference in the manner of spacing of cores; the transcriptionally active (yeast and HeLa) chromatins have spacings of variable length while the transcriptionally inactive (chicken erythrocyte) has a more regular spacing of cores.

Analysis of subunit organization in chicken erythrocyte chromatin.

Micrococcal nuclease digestion of intact chicken erythrocyte nuclei is shown to result in the formation of core nucleoprotein particles containing about 140 base pairs of DNA. These core particles, which are almost entirely devoid of histones f1 and f2c, are derived from transient nucleoprotein particles containing an average of approximately 180 base pairs of DNA. Oligomers of these latter particles may be isolated after brief nuclease digestion. The time course of digestion of these oligomers demonstrates the existence of "spacer" regions of more accessible DNA between core particles. Redigestion of purified monomer core nucleoprotein particles gives rise to both single-strand and double-strand DNA fragment patterns similar to those resulting from digestions of chromatin in situ. This observation indicates that the core particles we isolate are representative of nucleoprotein structures existing within the nucleus.