RESUMO
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.
Assuntos
Fator VIII/genética , Sequência de Aminoácidos , Anticorpos Monoclonais , Sequência de Bases , Clonagem Molecular , DNA/genética , Fator VIII/imunologia , Fator VIII/isolamento & purificação , Regulação da Expressão Gênica , Humanos , Rim/fisiologia , Oligodesoxirribonucleotídeos/síntese química , Fragmentos de Peptídeos/análise , Trombina , Cromossomo XRESUMO
Two DNA recombinant clones, shown by separate studies to contain DNA sequences homologous to the genes coding for the human blood coagulation Factors VIII and IX, were hybridized in situ to metaphases or prometaphases derived from patients with the fragile-X syndrome and from a normal control. The results of these experiments indicate that (i) both genes are located in the subtelomeric region of the long arm of the human X chromosome flanking the fragile site at Xq27.3, (ii) the resolution of this localization is approximately 0.5% the length of the human haploid genome, i.e., 1.8 X 10(7) bp, (iii) the linear order of loci within the above region is Factor IX-fragile site-Factor VIII-Xqter. Both the localization and the linear order of these loci have been confirmed by Southern blotting studies using the same molecular probes and a panel of rodent-human somatic cell hybrids known to have retained different segments of the human X chromosome. The findings described herein and the knowledge that Factor IX deficiency recombines freely with at least two loci of the G6PD cluster support our hypothesis that the chromosomal region which includes the fragile-X site is normally a region of high meiotic recombination.
Assuntos
Fator IX/genética , Fator VIII/genética , Síndrome do Cromossomo X Frágil/genética , Hemofilia A/genética , Hemofilia B/genética , Aberrações dos Cromossomos Sexuais/genética , Mapeamento Cromossômico , Ligação Genética , Glucosefosfato Desidrogenase/genética , Humanos , Hibridização de Ácido NucleicoRESUMO
We have constructed two simian virus 40 early replacement recombinants that have the coding sequences for hepatitis B virus surface antigen (HBsAg). One construction, LSV-HBsAg, has the coding region for HBsAg but not the portion encoding the putative pre-surface antigen leader. Transformed monkey kidney cells (COS) infected with this recombinant express large quantities of the characteristic partially glycosylated HBsAg molecule, which are assembled into 22-nm particles that appear similar to those produced by human liver cells infected with hepatitis B virus. This result indicates that the pre-surface antigen sequences are not required for the synthesis of HBsAg or its assembly into particulate structures. The second recombinant, LSV-HBpresAg, has the entire surface antigen gene, including the putative promoter and pre-surface antigen region. COS cells infected with this recombinant plasmid produce 40- to 50-fold less HBsAg than those infected with the LSV-HBsAg recombinant plasmid. RNA mapping studies suggest that the transcription of the HBsAg gene is initiated at more than one site, or alternatively, that RNA splicing of transcripts occurs in the pre-surface antigen region.
Assuntos
Genes Virais , Antígenos de Superfície da Hepatite B/genética , Transcrição Gênica , Animais , Linhagem Celular , Chlorocebus aethiops , DNA Recombinante , Vetores Genéticos , Peso Molecular , Splicing de RNA , RNA Viral/genética , RNA Viral/metabolismo , Vírus 40 dos Símios/genéticaRESUMO
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.
Assuntos
Cromossomos/análise , Elementos de DNA Transponíveis , Drosophila melanogaster/genética , Animais , DNA , Enzimas de Restrição do DNA , Hibridização de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico , Especificidade da EspécieRESUMO
DNA carrying inverted repeats was tested for transposition within the Drosophila genome. Five Bam HI segments containing related inverted repeats were isolated from D. melanogaster and analyzed by electron microscopy and restriction mapping. Southern blot experiments using single-copy flanking sequences as probes allowed the study of DNA arrangements at specific sites in the genomes of five closely related strains. We found that in some genomes the sequences with inverted repeats were present at a particular site, whereas in other genomes they were absent from this site. These results indicated that three of the sequences are transposable genetic elements. In one case we have purified the two corresponding DNA segments, with and without the sequence containing inverted repeats, thereby confirming the mobility of this sequence. These DNA elements were found to be distinct in two ways from copia and others previously described: first, they contain inverted terminal repeats, and second, they have a more heterogeneous construction.
Assuntos
Elementos de DNA Transponíveis , Drosophila melanogaster/genética , Animais , Sequência de Bases , DNA/genética , Hibridização de Ácido NucleicoRESUMO
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.
