The upstream region of the Xenopus laevis L14 ribosomal protein gene was deleted starting from the 5' extremity in order to define the promoter length necessary to express a linked reporter CAT gene. The functional analysis indicated that a sequence located between -63 and -49 from the capsite is important for an efficient promoter activity. Band shift and ExoIII protection assays evidenced the binding to this region of a factor, called XrpFI, present in the crude nuclear extract from X.laevis oocytes. Methylation interference analysis localized the contacts in the G residues belonging to a short box, 5' CTTCC 3', positioned between -53 and -49 from the capsite. An additional factor, XrpFII, makes contacts with the sequence 5'GCCTGTTCGCC 3' located between -27 and -17 from the capsite. The deletion mutant still containing this sequence is poorly transcribed, but resumes activity when a short fragment containing the binding site for factor XrpFI is cloned in an upstream position.

The availability of engineered serine proteases allows one to study the activation, substrate specificity and regulation of human coagulation and fibrinolytic activities. Human coagulation factor XI1 is composed of the protease catalytic region at the C-terminus, a hinge proline-rich region and regulatory domains at the N-terminus. From cDNA clones coding for factor XII, two DNA molecules were constructed, one being full length and the other being deleted of exons coding for the regulatory domains. Engineered factor-XI1 cDNA species were inserted by a homologous recombination technique into vaccinia viruses. which were used to infect the human hepatoma cell line HepG2. Two recombinant proteins were prepared from the culture media and identified by their antigenic properties and electrophoretic mobilities. The recombinant protein of larger size was identified as the full-length factor XI1 of 80 kDa and its specific activities and activation patterns, determined both by the coagulation and the amidolytic assays, are very similar to these of native human factor XII. The recombinant protein of smaller size was identified as a 319-amino-acid-deleted factor-XI1 protein of 32 kDa, containing only the entire protease region and part of the proline-rich hmge. This protein was expected to be the 'minimal' portion of Factor XI1 able to sustain protease activity, but unable to recognize substrates and surfaces necessary to activate the contact phase of coagulation. However, this 'minimal' Factor-XI1 protein displays a marked protease activity and, although lacking five regulatory domains of factor XLI, is bound and activated by negative charges and promotes coagulation with high efficiency.Events relevant to cellular and extracellular processes are regulated by the sequential enzymatic activation of proteins. One example of this is protein kinase activity leading to the regulation of important functions in the cell cycle and differentiation. Cascades of serine protease activities bring into effect and regulate extracellular processes of great importance to vertebrate organisms, such as blood coagulation, fibrinolysis and inflammation. The multiple and composite functions of serine proteases may be ascribed to structural domains able to recognize activating surfaces, inhibitors, cofactors and other serine proteases. The present study is aimed at producing and characterizing recombinant (r) human factor XI1 (FXII) proteins in order to clarify the functional role of the different domains composing human FXII.

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