Overcoming inhibition of antibody responses to a malaria recombinant vaccinia virus caused by prior exposure to wild type virus.

Pre-injection of mice with vaccinia virus inhibited the subsequent antibody response to a recombinant polypeptide expressed by vaccinia virus. The inhibition was overcome following additional challenges with recombinant vaccinia virus. This suggests that a potential disadvantage in vaccinia-immune individuals can be circumvented and may be outweighed by the advantages of the vector.

Partial deletion of the human host range gene in the attenuated vaccinia virus MVA.

The genome of a strongly attenuated vaccinia virus strain, MVA, was investigated by Southern blot and sequence analyses. Three major deletions, relative to the WR strain, were localized in MVA DNA. The deletions occurred near both ends of the viral genome and one of them affected a 55 K as well as the 32 K human host range gene. Although more than two thirds of the host range gene were eliminated from the MVA, the virus could still multiply in certain human cells.

Active gamma-carboxylated human factor IX expressed using recombinant DNA techniques.

Factor IX (Christmas factor), a vitamin K-dependent plasma protein made in the liver, functions in the middle phase of the intrinsic pathway of blood coagulation. A functional deficiency of factor IX underlies haemophilia B, a chromosome X-linked recessive disease for which the major therapeutic approach is replacement treatment using factor IX concentrates. The cloning and characterization of the gene for human factor IX would mean that human factor IX could be produced in greater yield and purity through using recombinant DNA techniques. We have now used a human factor IX cDNA clone, inserted into a vaccinia virus-derived vector, to infect human hepatoma cells which normally produce no factor IX, and mouse fibroblasts. Fully active factor IX was produced by the hepatoma cells, whereas the fibroblasts produced a protein less active than natural factor IX, even in the presence of high levels of vitamin K. Human factor IX is extensively post-translationally modified, and thus represents probably the most complex protein produced in active form by recombinant DNA techniques to date. Our study also illustrates the potential of vaccinia virus-based vectors for expressing significant amounts of complex, clinically useful proteins in eukaryotic cells, in addition to its already demonstrated usefulness for producing live recombinant vaccines.

Sequence specific cleavage of DNA by micrococcal nuclease.

Micrococcal nuclease is shown to cleave DNA under conditions of partial digestion in a specific manner. Sequences of the type 5'CATA and 5'CTA are attacked preferentially, followed by exonucleolytic degradation at the newly generated DNA termini. GC-rich flanking sequences further increase the probability of initial attack. Unexpectedly, long stretches containing only A and T are spared by the nuclease. These results, which were obtained with spared by the nuclease. These results, which were obtained with mouse satellite DNA and two fragments from the plasmid pBR22, do not support the previous contention that it is the regions of high At-content which are initially cleaved by micrococcal nuclease. This specificity of micrococcal nuclease complicates its use in experiments intended to monitor the nucleoprotein structure of a DNA sequence in chromatin.

DNA sequence of the constant gene region of the mouse immunoglobulin kappa chain.

The constant (C; ref. 3) gene segment of the immunoglobulin kappa light chain and about 1 kb upstream as well as downstream of the segment have been sequenced. The sequences of the C gene segment itself and parts of the upstream region were determined both in liver and in myeloma T DNA clones derived from the same mouse inbred strain. The sequences were identical, i.e. no somatic mutations were detected. Two sites in the region not coding for protein are discussed as possible targets of aberrant variable (V) gene translocations. Doublet frequencies were calculated in the approx. 2500 bp of the C region sequence reported in this paper and in the approx. 3400 bp of two rearranged V gene regions.

Nucleotide sequence of mouse satellite DNA.

The nucleotide sequence of uncloned mouse satellite DNA has been determined by analyzing Sau96I restriction fragments that correspond to the repeat unit of the satellite DNA. An unambiguous sequence of 234 bp has been obtained. The sequence of the first 250 bases from dimeric satellite fragments present in Sau96I limit digests corresponds almost exactly to two tandemly arranged monomer sequences including a complete Sau96I site in the center. This is in agreement with the hypothesis that a low level of divergence which cannot be detected in sequence analyses of uncloned DNA is responsible for the appearance of dimeric fragments. Most of the sequence of the 5% fraction of Sau96 monomers that are susceptible to TaqI has also been determined and has been found to agree completely with the prototype sequence. The monomer sequence is internally repetitious being composed of eight diverged subrepeats. The divergence pattern has interesting implications for theories on the evolution of mouse satellite DNA.

Functional and non-functional joining in immunoglobulin light chain genes of a mouse myeloma.

Leader, variable (V) and joining (J) gene segments, and adjacent regions of two rearranged alleles of the same kappa-chain producing mouse myeloma, comprising approximately 3,200 base pairs, have been sequenced. Sequence comparisons are reported. V-J joining in one of the alleles leads to a reading frame with a stop codon within the J-gene segments. Allelic exclusion is apparently realized in this tumour through the formation of such a non-functional allele.

A rearranged DNA sequence possibly related to the translocation of immunoglobulin gene segments.

A 5.3 kb EcoRI fragment (T3, abbreviations in ref. 2) has been cloned from DNA of a kappa light chain producing mouse myeloma. The fragment hybridizes to the k' flanking sequences of the J1 gene segment but not to C gene sequences of kappa light chain DNA. Restriction nuclease mapping and partial nucleotide sequencing showed that the fragment consists of sequences from the 5' side of the J1 and form the 3' side of a V gene segment, which apparently had been linked in a genomic rearrangement process. These rearranged flanking sequences are not the flanking sequences of the V and J gene segments which had been joined to form the two kappa light chain genes of the myeloma. Fragments with the hybridization properties of T3 have been found also in two other kappa and one lambda chain producing myelomas. The linking of flanking sequences in the myeloma genome is discussed with respect to the mechanism of recombination between V and J gene segments.

Comparative analysis of three guinea pig satellite DNA's by restriction nucleases.

The structures of guinea pig satellite DNAs I, II, and III have been analyzed by digestion with seven restriction nucleases. From the cleavage patterns it is obvious that the long-range periodicities in these three satellites differ rather characteristically Satellite I is fairly resistant to six nucleases and gives only a number of weak discrete bands which do not show a simple regularity. By the restriction nuclease from Arthrobacter luteus, however, it is cleaved extensively and yields very heterogeneous breakdown products. This is consistent with the high extent of divergence previously found for this satellite, e. g. by sequence analysis. Satellite II is almost completely resistant to all nucleases, indicative of a high degree of sequence homogeneity of this satellite. Satellite III is completely broken by the restriction nuclease from Bacillus subtilis into fragments which form a novel, highly regular series of bands in gel electrophoresis. The patterns show that the satellite is composed of tandem repeats ofapproximately 215 nucleotide pairs length, each repeat unit containing two cleavage sites for this nuclease. The data are consistent with the assumption that 30--40% of all cleavage sites have been eliminated by a random process. Satellite III DNA yields weak degradation patterns of the same periodicity with a number of other restriction nucleases. Cleavage sites for these nuclease are clustered on separatesmall segments of the satellite DNA. In this respect, the satellite is similar to others, notably the mouse satellite DNA. The three guinea pig satellites are examples of more general types of satellite structures also found in othe organisms. Similarities and differences to other satellites are discussed with special consideration to theories on the evolution of this class of DNA.

Nuclease cleavage of chromatin at 100-nucleotide pair intervals.

Digestion of mouse liver nuclei with DNase II leads to a novel cleavage pattern with a 100-nucleotide pair periodicity. From chromatin, this pattern or the standard 200-nucleotide pair repeat can be produced depending on the ionic conditions. The results are interpreted by assuming different conformational states of the nuclear material, including condensed and extended forms.