ABSTRACT
Human coagulation factor VIII:C has been purified approximately 5000-fold from commercial preparations with an average activity yield of 35%. Proteins of 92 kD and 77-80 kD enriched during purification are precipitated by a human serum polyclonal antibody which inhibits factor VIII:C activity. Evidence suggests that these polypeptides are linked by a calcium ion bridge. Partial amino acid sequence information from these proteins has been obtained from the intact polypeptides and from products of digestion with thrombin, endoproteinase lysC, or trypsin after citraconylation. An oligonucleotide probe designed from one of the amino acid sequences was used to isolate a partial genomic clone from a human 4X chromosome library in bacteriophage lambda. The genomic segment was used to isolate two cDNA molecules encompassing the entire human kidney factor VIII:C mRNA. Biologically active factor VIII:C has been produced in a mammalian cell line utilizing a complete cDNA construction.
Subject(s)
Factor VIII/genetics , Amino Acid Sequence , Antibodies, Monoclonal , Base Sequence , Cloning, Molecular , DNA/genetics , Factor VIII/immunology , Factor VIII/isolation & purification , Gene Expression Regulation , Humans , Kidney/physiology , Oligodeoxyribonucleotides/chemical synthesis , Peptide Fragments/analysis , Thrombin , X ChromosomeABSTRACT
We have analyzed the construction of the members of the foldback (FB) family of transposable elements in Drosophila by detailed restriction analysis, cross hybridization, electron microscopy, in situ hybridization and nucleotide sequence determination. The members are heterogeneous, with both the inverted terminal repeats and the total element sizes extremely variable. Nevertheless, the ends of the inverted repeats represent closely conserved sequences, similar for all members. Sequence analysis of one of the FB elements revealed an unusual constriction. There are scattered multiple copies of a 10 bp sequence near the inverted repeat termini; 300 bp from the end this sequence is expanded to a 20 bp repeat, and 500 bp from the end it is again expanded to a 31 bp repeat. A large part of the inverted repeats consists of contiguous tandem repeats of this 31 bp sequence. We also find two differences between the two copies of the inverted repeat, one of which involves an intact copy of the 10 bp sequence mentioned above. Sequence analysis of a corresponding DNA segment without this transposable element shows that insertion generates a 9 bp duplication at the target site. In situ hybridizations to polytene chromosomes show about 30 widely scattered positions with homology. Comparison of the hybridization patterns for three strains shows significant interstrain and intrastrain differences in chromosomal locations.
Subject(s)
Chromosomes/analysis , DNA Transposable Elements , Drosophila melanogaster/genetics , Animals , DNA , DNA Restriction Enzymes , Nucleic Acid Hybridization , Repetitive Sequences, Nucleic Acid , Species SpecificityABSTRACT
The replication of extrachromosomal rDNA molecules from the macronucleus of Tetrahymena was studied by electron microscopy. Replication begins in the center of the palindromic molecule and proceeds by means of bidirectional fork movement toward the free ends of the molecule.