CD4-affinity purification of recombinant and native HIV gp120 and comparison of the affinity constants for the receptor.

Soluble CD4-immunoglobulin chimeric proteins were covalently attached to CNBr-activated Sepharose. This affinity matrix was used to establish a powerful new method to isolate different species of the HIV external glycoprotein gp120 from cell-free culture supernatants. Recombinant gp120 was expressed in Baculovirus-infected insect cells and isolated from cell-free culture supernatants. The recombinant protein has an apparent molecular mass of 130 kDa. These two gp120 species were shown to be of identical molecular size after complete deglycosylation achieved by endoglycosidase treatment, and they bound to CD4-H gamma l with the same binding constant, that was reported for native forms of gp120 and CD4. Thus the different glycosylation of gp120 does not influence its affinity to CD4 and the gp120-CD4 complex can be reversibly dissociated.

The androgen-dependent rat prostatic binding protein: comparison of the sequences in the 5' part and upstream region of the C1 and C2 genes and analysis of their transcripts.

The complete gene encoding the polypeptide C1 of the complex androgen-controlled prostatic binding protein was isolated from a rat genomic library. A new genomic fragment (C2B) containing only the 5' part of a C2-related gene was also purified. The segments containing exon 1 and a large part of the adjacent sequences were analysed and compared with the corresponding region of the C2A gene which has been completely sequenced previously. The high structural similarity extending over a large part of all three genomic fragments suggests the duplication of a common ancestral gene, followed by a more recent duplication of the C2-coding region. However, since the structural similarity upstream of position -150 between C2A and C2B abruptly disappears and no transcripts specific for the C2B region can be detected in prostate RNA, we propose that at a later stage in evolution the C2B region was disrupted and inactivated. Despite the common origin and the similar regulation of the two active genes, C1 and C2A, the only obvious conserved structural element is the homopurine stretch located at position -400, although sequence motifs resembling steroid hormone response elements are present at several locations.

Mutation of conserved N-glycosylation sites around the CD4-binding site of human immunodeficiency virus type 1 GP120 affects viral infectivity.

Infection by the human immunodeficiency virus type 1 (HIV-1) is initiated through interaction of its exterior envelope glycoprotein gp120 with the CD4 receptor on target cells. To address the possible role of N-glycosylation of HIV-1 gp120 in binding CD4, we mutated different conserved N-glycosylation site Asn-residues in the vicinity of the putative CD4 binding site, as single mutations or in combinations. Authentic and mutant gp120 proteins were produced using the baculovirus expression system. All mutant proteins were produced and secreted at similar levels and could be immunoprecipitated with an HIV(+)-serum. Furthermore, all glycosylation mutants retained the full capacity to bind CD4 except for a triple mutant which showed reduced binding. Different gp120 mutant genes were then introduced in an infectious proviral DNA clone. Upon transfection of MT-2 cells, the authentic HIV-1 clone induced maximal virus production after 6 days. In the case of the triple glycosylation mutant, comparable virus production was first reached after a delay of about 12 days. Moreover, in contrast to native HIV, the mutant virus induced no typical multinucleated giant cells. These results suggest that the attached carbohydrates around the CD4-binding site of gp120, may contribute to the generation of this protein domain required for high affinity receptor interaction.

Rat prostatic binding protein: the complete sequence of the C2 gene and its flanking regions.

The complete sequence (2879 bp) of the androgen-controlled rat prostatic binding protein C2 gene and 1023 bp of the 5'- and 2127 bp of the 3'-flanking regions have been determined. The gene contains three exons (93, 203 and 147 bp) and two introns (1630 and 806 bp). It is flanked by two homopurine-homopyrimidine stretches of 55 and 131 nucleotides respectively, located at positions -405 and 4151. These sequences are remarkably sensitive towards S1-nuclease, indicating an altered DNA conformation under superhelical stress. Several palindromes and dyad structures are observed in the 5'-upstream region of the gene and at position -457, and 80% homology to the consensus sequence of a glucocorticoid receptor binding site is found.

The nucleotide sequence of cDNA complementary to the C1 component of rat prostatic binding protein.

The mRNA for component C1 of rat prostatic binding protein has been cloned and characterized. A partially purified mRNA fraction for this complex protein was reverse-transcribed into double-stranded cDNA and cloned into the PstI site of plasmid pBR 322. The 426-base-pair insert of the recombinant plasmid pC1A75 was completely sequenced. The coding region corresponds precisely to the 88 amino acid residues of C1 and in addition contains the information of a signal peptide of 23 residues. The 5' non-coding region counts only 19 nucleotides and is incomplete but the 3'-terminal non-coding part of 60 nucleotides extends into the poly(A) tail. Sequence analysis of other C1-positive clones indicates the presence of sequence rearrangements which must have occurred during the cloning procedure. Possible mechanisms for the generation of these cloning artefacts are discussed.

Mapping of rat prostatic binding protein genes C1, C2, and C3 to rat chromosome 5 by in situ hybridization.

Rat prostatic binding protein genes C1, C2, and C3 were mapped on rat chromosome 5 by in situ hybridization on rat peripheral blood chromosome preparations using three different cDNA probes. Of the grains detected, 15.9%, 25.2%, and 19.6%, respectively, mapped to chromosome 5. For each probe, the label was predominantly located on 5q31, but for C2 and C3 an additional site on 5q21 was found. The results suggest that three genes coding for the different polypeptide chains of rat prostatic binding protein map to the same chromosome and presumably to the same chromosome band.

Comparison of the 5' upstream putative regulatory sequences of three members of the alpha 2u-globulin gene family.

We have isolated and characterized seven members of the alpha 2u-globulin gene family from a rat genomic library. The 5' upstream region (up to 1250 base pairs starting from the EcoRI site in exon 2) of three clones was sequenced. The major transcriptional start points were located 25 base pairs downstream from the 'TATA' box. A very high degree of homology was observed over the entire studied region. Two of the examined genes displayed structural features which suggest that their expression may be impeded. A high degree of homology was observed between the promotor regions of alpha 2u-globulin and those of the major urinary protein (MUP) multigene family of the mouse. A remarkable feature is the variable length of an A-rich region between the putative 'CAAT' and the 'TATA' consensus sequences. The size of this region differs markedly between MUP and alpha 2u-globulin and between different members of the alpha 2u-globulin gene family. Comparison of the alpha 2u-globulin promotor with the corresponding region of other androgen-dependent genes (the C1, C2 and C3 subunits of prostatic steroid binding protein) reveals the presence of an A-rich region of homology located approximately 378 base pairs upstream from the cap site in the alpha 2u-globulin genes. This region compares well with a sequence of putative enhancer function previously demonstrated in the alpha-fetoprotein promotor and in the immunoglobulin heavy chain promoter.

A single 12.5-kilobase androgen-regulated mRNA encoding multiple proline-rich polypeptides in the ventral prostate of the rat.

Synthetic 32P-labeled oligonucleotides have been used to identify the prostatic proline-rich polypeptide (PRP) mRNA which has partially been characterized. The 14-mer d(G-G-T-T-C-T-G-C-A-T-A-A-T-G) complementary to the coding sequence for His-Tyr-Ala-Glu-Pro, a sequence element occurring in all 38-residue PRP variants, hybridizes specifically with a 12.5-kilobase mRNA which is clearly androgen-controlled. This oligonucleotide was used as an efficient primer for the construction of a PRP-specific lambda gt10 cDNA library. The nucleotide sequence of the inserts from several recombinant clones has been determined. This structural analysis revealed a PRP mRNA encoding a large precursor containing a number of tandemly repeated units. Each repeat codes for a sequence of 100 amino acids in which the highly conserved PRP sequence is embedded. From this polyprotein the large number of PRP variants must be generated by a post-translational processing mechanism which is still unknown. The high degree of conservation of both nucleotide and amino acid sequence in the entire unit also indicates that the PRP gene(s) likely evolved by multiplication of a 300-base pair ancestral DNA sequence. This has resulted in a noninterrupted repetitive DNA coding segment which is detected at the genomic level.