A human liver cDNA library enriched for full-length clones was screened for plasminogen cDNA using a synthetic 24nucleotide probe derived from a reported partial cDNA sequence. 12 positive clones were identified and one of these was characterized in detail. The 2.7 kb insert contains the complete coding region. At 5 positions, it gives residues different from those reported in a previous amino acid sequence analysis of the protein. The present results show an extra IIe at position 65. Gln instead of Glu at positions 53 and 342, Asn at position 88 instead of Asp, and Asp at position 453 rather than Asn. In the 3'-non-coding region an extension of 29 bases is found which does not contain any structure compatible with a known polyadenylation signal. Instead, the consensus signal AATAAA is placed at a distance of 46 bases upstream of the poly(A)-tail.

Certain strains of group B streptococci express a cell surface protein that binds IgA and acts as a virulence factor. This IgA receptor is referred to here as protein Bac. The gene for protein Bac was cloned and expressed in Escherichia coli, and the complete nucleotide sequence was determined. The deduced amino acid sequence of 1134 residues includes a signal sequence of 37 amino acids and a putative membrane anchor region at the C-terminal end. The processed form of the receptor, 1097 residues, has a calculated molecular weight of 123,786. There are no cysteines in protein Bac, suggesting a fibrillar structure. The C-terminal half of the protein includes a 90 residues long region with a novel type of periodic structure, the "XPZ motif", in which every third amino acid is proline. Unlike other bacterial immunoglobulin-binding proteins, there are no long repeats in protein Bac. Clones which express only part of the protein Bac gene were used to show that IgA-binding takes place in the N-terminal part of the molecule. Protein Bac was originally described as an antigen called beta, but N-terminal fragments that bind IgA do not react with a reference serum against the beta antigen. These and other data indicate that protein Bac can be divided into two regions with different functions: an N-terminal IgA-binding region and a C-terminal region corresponding to the beta antigen. The IgA-binding region of protein Bac does not show any homology to protein Arp, the IgA receptor from group A streptococci, although these receptors have similar binding properties. This indicates that convergent evolution has favored the appearance of these two structurally different streptococcal IgA receptors.

Many strains of Streptococcus pyogenes are known to express a receptor for IgA. The complete nucleotide sequence of the gene for such a receptor, protein Arp4, has been determined. The deduced amino acid sequence of 386 residues includes a signal sequence of 41 amino acids and a putative membrane anchor region, both of which are homologous to similar regions in other streptococcal surface proteins. The processed form of the IgA receptor has a length of 345 amino acids and a calculated molecular weight of 39544. The N-terminal sequence of the processed form is different from that previously found for a similar IgA receptor isolated from a S. pyogenes strain of type M60. The sequence of protein Arp4 shows extensive homology to the C-terminal half of streptococcal M proteins, but not to the streptococcal IgG receptor protein G or staphlyococcal protein A. Apart from the membrane anchor, this homology includes a sequence of 119 amino acid residues containing three repeated units and a 54-residue sequence without repeats. The protein expressed in Escherichia coli is found in the periplasmic space, in which it constitutes the major protein. Protein Arp4 is the first example of a surface protein that has both immunoglobulin-binding capacity and structural features characteristic of M proteins.

The gene for protein D, a membrane-associated protein with specific affinity for human immunoglobulin D, was cloned from a nontypeable strain of Haemophilus influenzae. The gene was expressed in Escherichia coli

Three different size classes of cDNA clones coding for the &subunit of human alcohol dehydrogenase (ADH) were characterized from a human liver cDNA library. Clones were identified by hybridization with synthetic oligodeoxyribonucleotides.A total of 2530 nucleotides were determined, covering an ADH-coding region of 1122 nucleotides, a preceding 72-nucleotide segment and 3 types of 3'-non-coding region. The coding nucleotide sequence is in full agreement with the amino acid sequence of the &subunit. Of 8 clones identified, 6 had a short, 213-nucleotide 3'-non-coding region; 1 an intermediate, 590-nucleotide 3'-region; and 1 a long, 1330-nucleotide 3'-region. In addition, 2 unused polyadenylation signals were found. These results suggest that human liver /I-ADH mRNAs occur in several size classes, and that in addition to the consensus sequence AATAAA further signals are important for 3'-end formation.Alcohol dehydrogenase cDNA

cDNA clones corresponding to two alleles of the ADH3 locus were identified by hybridization with synthetic oligodeoxyribonucleotides specific for class I human liver alcohol dehydrogenase. Sequences were determined for a 1457‐nucleotide cDNA, covering the whole γ2‐coding region, and a 1224‐nucleotide cDNA, including the region coding for amino acid residues 53–374 of the γ1 subunit. Two amino acid replacements between the γ1 and γ2 subunits were identified. At position 349, isoleucine in γ1 instead of valine in γ2 is a conservative exchange of a superficial residue which has been ascribed no special importance. The other exchange, at position 271, arginine in γ1 and glutamine in γ2, explains differences in enzyme properties. Electrophoretically, it is consistent with the less cathodic mobility of the γ2 subunit. Functionally, the location of the exchange at the surface of the coenzyme‐binding pocket may influence the dissociation of the reduced coenzyme.

Protein D is an immunoglobulin D-binding membrane protein exposed on the surface of the gram-negative bacterium Haemophilus influenzae. Results reported here indicate that protein D is a Jipoprotein. The protein is apparently synthesized as a precursor with an 18-residue-long signal sequence modified by the covalent attachment of both ester-linked and amide-linked palmitate to the cysteine residue, which becomes the amino terminus after cleavage of the signal sequence. Globomycin inhibited maturation of protein D in H. influenzae, implying that protein D is exported through the lipoprotein export pathway. A mutant expressing a protein D lacking the cysteine residue was constructed by oligonucleotide site-directed mutagenesis. The mutated protein D molecule was not acylated and partitioned in the aqueous phase after Triton X-114 extraction of intact bacteria, unlike native and recombinant protein D, which partitioned in the detergent phase. The nonacylated protein D molecule was localized to the periplasmic space of Escherichia coli. The hydrophilic protein D molecule will be used in investigations concerning its ability to function as a vaccine component.

Proline-rich regions have been identified in many surface proteins of pathogenic streptococci and staphylococci. These regions have been suggested to be located in cell wall-spanning domains and/or to be required for surface expression of the protein. Because little is known about these regions, which are found in extensively studied and biologically important surface proteins, we characterized the proline-rich region in one such protein, the ␤ protein of group B streptococci. The proline-rich region in ␤, designated the XPZ region, has a proline at every third position, and the sequence is highly periodic in other respects. Immunochemical analysis showed that the XPZ region was not associated with the cell wall but was exposed on the bacterial surface. Moreover, characterization of a ␤ mutant lacking the XPZ region demonstrated that this region was not required for surface expression of the ␤ protein. Comparison of the XPZ region in different ␤ proteins showed that it varied in size but always retained the typical sequence periodicity. Circular dichroism spectroscopy indicated that the XPZ region had the structure of a polyproline II helix, an extended and solvent-exposed structure with exactly three residues per turn. Because of the three-residue sequence periodicity in the XPZ region, it is expected to be amphipathic and to have distinct nonpolar and polar surfaces. This study identified a proline-rich structure with unique properties that is exposed on the surface of an important human pathogen.