Reevaluation of total, free, and bound protein S and C4b-binding protein levels in plasma anticoagulated with citrate or hirudin.

The levels of total, free, and bound protein S and C4BP were determined using enzyme-linked immunosorbent assays (ELISAs) in plasma samples (8 males and 8 females) that were individually subjected to immunoadsorption studies in which "free protein S" was defined as that not adsorbed by anti-C4BP antibody-Sepharose column and "free C4BP" as that not adsorbed by anti-protein S antibody-Sepharose. Bound species were calculated as the difference between total and free species. Free protein S (131 nmol/L) averaged 38% of total protein S (346 nmol/L) and free C4BP (37 nmol/L) averaged 14% of total C4BP (264 nmol/L) in plasma. There was an excellent correlation between bound protein S and bound C4BP with 1:1 molar stoichiometry and a good correlation between free protein S antigen and protein S anticoagulant activity. It appears that free protein S is a necessary consequence of the molar excess of protein S over C4BP. C4BP beta chain antigen levels, measured using a new ELISA, averaged 218 nmol/L, a value indistinguishable from the molar concentrations of bound protein S (215 nmol/L) and bound C4BP (227 nmol/L). The free C4BP beta chain antigen was only 4 nmol/L compared with 131 nmol/L free protein S. These results suggest that free C4BP in plasma predominantly lacks the beta chain, that almost all C4BP capable of binding protein S is associated with protein S, and that the plasma levels of oligomeric forms of C4BP containing a beta chain (alpha 7 beta and alpha 6 beta) that bind protein S directly and stoichiometrically regulate free protein S levels.     

Complete primary structures of the E beta chain and gene of the mouse major histocompatibility complex.

Using the cross-hybridization with plasmid pDC beta-1, containing the cDNA coding for the DC beta chain of the human major histocompatibility complex class II molecules, we have cloned and subjected to sequence analysis both the cDNA and genomic gene for the E beta chain of the BALB/c (d haplotype) mouse. The nucleotide sequences of the cDNA and genomic DNA clones permitted us to deduce the entire primary structure of the E beta chain and the complete exon-intron structure of the E beta gene. Unlike alpha chain genes that contain five exons, the E beta gene consists of six exons corresponding to the six functional domains--the leader, beta 1 and beta 2 domains, transmembrane peptide, intracytoplasmic peptide, and 3' untranslated region. In addition, two short blocks of sequences common to alpha and beta chain genes were identified in the 5' flanking regions. We propose that these sequences are involved in the coordinate expression of alpha and beta chains.     

Separation of sequences defining basal expression from those conferring alpha gene recognition within the regulatory domains of herpes simplex virus 1 alpha genes.

The genes of herpes simplex virus 1 form three major groups--alpha, beta, and gamma--whose expression is coordinately regulated and sequentially ordered in a cascade fashion. To determine how the infected cell differentiates between these gene groups, alpha-regulated chimeric genes were constructed in earlier studies by fusing the structural sequences of the thymidine kinase (TK) gene, a beta gene, to the 5' noncoding sequences of alpha genes. These studies showed that (i) one or more structural components of the virion act in trans to increase alpha gene expression and (ii) the 5' noncoding sequences of alpha genes contain cis-acting domains that promote gene expression and confer alpha-gene regulation. These two domains could be moved independently, but the regulatory domain required a promoter for its function. We report here the properties of three sequences containing features common to the regulatory regions of all alpha genes. Sequence 1, containing (G + C)-rich inverted repeats, increased the basal level of TK expression when fused 5' to either the alpha gene 4 promoter or the truncated beta TK promoter. The effect was to some extent orientation dependent. Moreover, sequence 1 restored beta regulation to the truncated beta TK promoter but did not confer alpha-specific regulation on any of the chimeric genes tested. Sequences 2 (49 base pairs) and 3 (29 base pairs), containing an (A + T)-rich homolog from alpha gene 27 and alpha gene 0, respectively, restored alpha-specific regulation to the alpha promoter gene but only sequence 2 conferred alpha regulation on the truncated beta promoter gene. Our results indicate that (i) in natural beta TK the promoter and regulatory domains overlap, (ii) sequence 1 determines basal level of expression and substitutes for a promoter component that is essential for beta but not alpha regulation, and (iii) conversion of a gene with a promoter into an alpha gene requires two elements. Sequence 2 may contain both whereas sequence 3 contains only one.     

Expression of fully functional tetrameric human hemoglobin in Escherichia coli.

Synthetic genes encoding the human alpha- and beta-globin polypeptides have been expressed from a single operon in Escherichia coli. The alpha- and beta-globin polypeptides associate into soluble tetramers, incorporate heme, and accumulate to greater than 5% of the total cellular protein. Purified recombinant hemoglobin has the correct stoichiometry of alpha- and beta-globin chains and contains a full complement of heme. Each globin chain also contains an additional methionine as an extension to the amino terminus. The recombinant hemoglobin has a C4 reversed-phase HPLC profile essentially identical to that of human hemoglobin A0 and comigrates with hemoglobin A0 on SDS/PAGE. The visible spectrum and oxygen affinity are similar to that of native human hemoglobin A0. The recombinant protein shows a reduction in Bohr and phosphate effects, which may be attributed to the presence of methionine at the amino termini of the alpha and beta chains. We have also expressed the alpha- and beta-globin genes separately and found that the expression of the alpha-globin gene alone results in a marked decrease in the accumulation of alpha-globin in the cell. Separate expression of the beta-globin gene results in high levels of insoluble beta-globin. These observations suggest that the presence of alpha- and beta-globin in the same cell stabilizes alpha-globin and aids the correct folding of beta-globin. This system provides a simple method for expressing large quantities of recombinant hemoglobin and allows facile manipulation of the genes encoding hemoglobin to produce functionally altered forms of this protein.     

Unusual sequence homology at the 5-ends of the developmentally regulated beta A-, beta C-, and gamma-globin genes of the goat.

We have constructed a recombinant library of goat DNA and have isolated clones containing the beta A-, beta C-, and gamma-globin genes and a presumptive embryonic globin gene. These genes are switched on and off during development and, thus, provide a model system for the study of gene regulation. To identify regions that may be involved in this switch, we determined the sequence of the areas surrounding the 5' ends of the beta A-, beta C- and gamma-globin genes. Surprisingly, the sequences of the beta A-, and beta C-globin genes are identical, beginning with the translation initiation codon and extending 131 base pairs (bp) in the 5' direction. At this point, two nucleotide differences are seen and only six additional differences are encountered when the sequences are extended 144 bp further in the 5' direction. Furthermore, the nucleotide sequence of the 5' end of the gamma-globin gene is very similar to those of the beta A-0, and beta C-globin genes. Only three nucleotide differences are found in the gamma-globin gene within the 131-bp region in which the beta A- and beta C-globin genes are identical. We conclude that this identical region cannot contain regulatory signals that mediate the switch from beta C to beta A expression.     

Studies on murine Ss protein: demonstration that S region encodes structural gene for fourth component of complement.

Although genes controlling the expression of certain complement components have been shown to be linked to the major histocompatibility complex of several species, the structural genes that encode these molecules have been more difficult to map. In this study, the three constitutive polypeptide chains of the fourth component of murine complement (C4) (alpha, beta, and gamma) were isolated from 14 different inbred strains and compared by peptide mapping on analytical sodium dodecyl sulfate gels. The peptide patterns of the alpha and gamma subunits appeared to be nearly identical, but two distinctly different patterns were observed for the C4 beta chain. This structural variant was mapped to the S or G region and, as such, provides direct evidence that a structural gene for a complement component is encoded within a major histocompatibility complex.     

Structural organization of the DR subregion of the human major histocompatibility complex.

Two clusters of overlapping cosmid and lambda phage clones comprising 205 kilobases (kb) have been isolated from the DR subregion of the human major histocompatibility complex from a DR4 haplotype. A single DR alpha and three DR beta genes were identified. In one cluster (135 kb), the DR alpha gene is 90 kb distant from the DR beta gene encoding a molecule that carries the MT3 serological specificity. In the second cluster (70 kb), the DR beta gene determining the DR4 specificity is located 22 kb apart from a DR beta pseudogene (DR beta psi). A 3- to 4-kb sequence located at the 5' end of the DR beta (MT3) gene is common to all three DR beta-chain genes. In addition, three more copies of this sequence are spaced between the DR alpha and the DR beta (MT3) genes in the first cluster and one of these, at least, is associated with a DR beta 1 exon, suggesting that additional genes could be encoded in this region and that multiple duplication events have led to its evolution.     

Decline in the expression of C4 binding protein alpha-chain gene during ageing of the rat liver

It appears that consistent changes in the levels of activity of a small cohort of genes(probably less than 1% of all active genes)occur in all mammalian cells during ageing. We have studied this phenomenon in rat liver using an optimised form of differential display. During this investigation we observed one gene which exhibited a decline in expression in livers from young adult (6 months) to aged adult (24 months) animals. The differential expression of this gene was confirmed by single strand conformational polymorphism (SSCP) gel analysis and Northern blotting. Densitometry of the latter indicated that there was adecline of 35% in its expression with age. Characterisation of the isolated PCR fragment demonstrated it to code for the alpha subchain of the complement 4 binding protein (C4BP). The C4BP is a key regulatory protein of the complement system and this observation therefore indicates that a decline in the efficiency of the complement system may be an important factor in the overall decline in immune function that has been observed during ageing. This revised version was published online in July 2006 with corrections to the Cover Date.     

Differentiation between alpha promoter and regulator regions of herpes simplex virus 1: the functional domains and sequence of a movable alpha regulator.

The herpes simplex virus genome consists of at least three groups of genes--alpha, beta, and gamma--whose expression is coordinately regulated and sequentially ordered in a cascade fashion. We have established that the elements involved in regulation of alpha genes are a sequence that promotes gene expression and a sequence that confers alpha regulation on the gene by responding to trans-acting regulatory signals. The domains of these sequences were mapped by determining the regulation of thymidine kinase (TK) in L cells converted to TK+ phenotype by chimeric TK indicator genes. The chimeric genes were constructed from appropriate portions of the TK gene fused to donor sequences derived from the 5' nontranscribed and nontranslated leader portions of the viral alpha gene 4. The results were as follows. (i) The natural beta TK indicator extending 5' up to -80 and the chimeric alpha TK extending 5' up to -110 both converted cells to TK+ phenotype but were not regulated. (ii) A segment of the regulator region of the alpha gene 4, extending 5' from position -110, confers inducible alpha-type regulation when fused to the nonregulated but expressible beta TK indicator described above. (iii) The extent of gene induction appears to hinge on the size of the regulatory region inserted into the chimeric gene and correlates with the presence of repeated consensus sequences and G+C-rich inverted repeats in the regulatory region of the alpha gene 4 and other alpha genes.     

Expression of haptoglobin-related protein and its potential role as a tumor antigen.

These studies describe the detection of a haptoglobin species, its characterization as the HPR gene product, and its association with both pregnancy and neoplasia. Previous work showed that the early recurrence of human breast cancer correlated with immunohistochemical staining with a commercial antiserum ostensibly directed against pregnancy-associated plasma protein A (PAPP-A). Use of this antiserum to guide purification of the putative antigen led to the present identification and purification of a strongly immunoreactive protein species distinct from PAPP-A that was present in the plasma of pregnant women at term. Unlike PAPP-A, a homotetramer of 200-kDa polypeptides, the immunoreactive protein consists of a light (alpha) chain (16.5 kDa) and a heavy (beta) chain (40 kDa); protein microsequencing of the beta chain showed it to be a member of the haptoglobin family. The alpha chain of this haptoglobin species differs from ordinary haptoglobin 1 and 2 alpha chains both structurally and immunologically and represents the product of the HPR gene, haptoglobin-related protein (Hpr), since (i) the apparent molecular mass is the same as that predicted for Hpr alpha chain, (ii) the peptide map differs from that of haptoglobin 1 in a manner predicted by the HPR nucleotide sequence, (iii) monospecific antibodies that react with epitopes shared by the unique alpha chain and a synthetic peptide derived from the HPR nucleotide sequence do not detect these epitopes in either haptoglobin 1 or 2, and (iv) sequences of alpha-chain peptides were consistent with this identification, excluding haptoglobin 1 but not haptoglobin 2. The immunohistochemical reactivity of antibodies raised to the synthetic Hpr peptide is similar to that of anti-PAPP-A. Moreover, staining of neoplastic breast tissue is abolished by preincubation with purified Hpr.     

Cloning of cDNA coding for the beta chain of human complement component C4b-binding protein: sequence homology with the alpha chain.

The major form of complement component C4b-binding protein, a regulator of the complement system, is composed of seven identical 70-kDa alpha chains, each containing a binding site for the complement protein C4b. We recently showed that C4b-binding protein also contains a unique 45-kDa beta chain. It is disulfide-linked to the central core and contains a binding site for the vitamin K-dependent protein S. We have now isolated and characterized full-length cDNA clones for the beta chain. In addition, 57% of the structure was determined by protein sequencing of tryptic and chymotryptic peptides. Two clones, A8 and C1, isolated from different libraries were sequenced. Except for a deleted triplet encoding Ala-3 in clone A8, the two clones were identical and coded for a leader sequence of 17 amino acids and a mature protein of 235 amino acids (including Ala-3). By N-terminal amino acid sequencing, the Ala-3 heterogeneity was confirmed and a third beta-chain species starting at Glu-4 was identified. The beta chain contains five potential N-linked glycosylation sites, and endoglycosidase digestion suggested that the beta chain contained multiple complex carbohydrate side chains. Northern blot analysis of human liver mRNA, using the beta-chain cDNA as the probe, demonstrated a major mRNA species of approximately 1.0 kilobase. From the N terminus, the beta chain contains three tandem repeat units (60 amino acids long) that are homologous to those present in the alpha chain. The C-terminal region, which was unrelated to the tandem repeats, demonstrated sequence similarity with the corresponding region of the alpha chain. In both alpha and beta chains these regions contain two cysteine residues that probably form the interchain disulfide bridges.     

Two genes encoding steroid 21-hydroxylase are located near the genes encoding the fourth component of complement in man.

Two genes encoding steroid 21-hydroxylase [21-OHase; steroid 21-monooxygenase; steroid, hydrogen-donor: oxygen oxidoreductase (21-hydroxylating); EC 1.14.99.10], a cytochrome P-450 enzyme, have been located within the HLA major histocompatibility complex. Congenital adrenal hyperplasia due to 21-OHase deficiency is a common inherited disorder of cortisol biosynthesis which is in genetic linkage disequilibrium with certain extended HLA haplotypes. These haplotypes include characteristic serum complement allotypes. A series of cosmid clones was isolated from a human genomic library by using a probe encoding part of the fourth component of complement, C4. These clones also hybridized with a probe encoding most of human 21-OHase. Restriction mapping and hybridization analysis showed that there are two 21-OHase genes, each located near the 3' end of one of the two C4 genes. Hybridization with probes specific for the 5' and 3' ends of the 21-OHase gene showed that the 21-OHase and C4 genes all have the same orientation. The 21-OHase genes 3' to C4A and C4B carry T aq I fragments of 3.2 and 3.7 kilobases (kb), respectively. Both of these fragments are found in genomic DNA of most individuals. In DNA from an individual with the severe, "salt-wasting" form of 21-OHase deficiency who was homozygous for HLA-A3;Bw47;C4A*1;C4B*Q0(null); DR7, the 3.7-kb Taq I fragment is absent, whereas hormonally normal individuals homozygous for HLA-A1;B8;C4A*Q0;C4B*1;DR3 do not carry the 3.2-kb Taq I fragment. These data suggest that the 21-OHase "B" gene (3.7-kb Taq I fragment) is functional, but the 21-OHase "A" gene (3.2-kb Taq I fragment) is not.     

Molecular mapping of the human major histocompatibility complex by pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis and "cosmid walking" have been used to establish a molecular map of the human major histocompatibility complex (MHC). We have isolated approximately equal to 230 kilobases (kb) of genomic DNA in overlapping cosmid clones covering the genes for the second and fourth components of complement (C2 and C4, respectively), factor B, and steroid 21-hydroxylase, and approximately equal to 82 kb of genomic DNA surrounding the genes for the tumor necrosis factors alpha and beta. Single-copy hybridization probes isolated from these cosmid clusters and probes for the known MHC gene loci were hybridized to Southern blots of genomic DNA that had been digested with infrequently cutting restriction endonucleases and separated on pulsed-field gels. The data obtained allowed the construction of a long-range genomic restriction map and indicated that the MHC spans 3800 kb. This map orients the MHC class III gene cluster with respect to the DR subregion; the C2 gene is on the telomeric side of the 21-hydroxylase B gene. In addition we have defined the positions of the genes for the tumor necrosis factors alpha and beta in the human MHC. Genes for the alpha chain of DR and 21-hydroxylase B are separated by at least 300 kb, while the distance between the genes for C2 and tumor necrosis factor alpha is 390 kb. The HLA-B locus lies approximately equal to 250 kb on the telomeric side of the tumor necrosis factor genes.     

Characterization of bone marrow laminins and identification of alpha5-containing laminins as adhesive proteins for multipotent hematopoietic FDCP-Mix cells

Laminins are extracellular matrix glycoproteins that influence the phenotype and functions of many types of cells. Laminins are heterotrimers composed of alpha, beta, and gamma polypeptides. So far five alpha, three beta, and two gamma polypeptide chains, and 11 variants of laminins have been proposed. Laminins interact in vitro with mature blood cells and malignant hematopoietic cells. Most studies have been performed with laminin-1 (alpha1beta1gamma1), and its expression in bone marrow is unclear. Employing an antiserum reacting with most laminin isoforms, we found laminins widely expressed in mouse bone marrow. However, no laminin alpha1 chain but rather laminin alpha2, alpha4, and alpha5 polypeptides were found in bone marrow. Our data suggest presence of laminin-2 (alpha2beta1gamma1), laminin-8 (alpha4beta1gamma1), and laminin-10 (alpha5beta1gamma1) in bone marrow. Northern blot analysis showed expression of laminin alpha1, alpha2, alpha4, and alpha5 chains in long-term bone marrow cultures, indicating upregulation of laminin alpha1 chain expression in vitro. Laminins containing alpha5 chain, in contrast to laminin-1, were strongly adhesive for multipotent hematopoietic FDCP-mix cells. Integrin alpha6 and beta1 chains mediated this adhesion, as shown by antibody perturbation experiments. Our findings indicate that laminins other than laminin-1 are functional in adhesive interactions in bone marrow.     

(Alpha)alpha 5.3: a novel alpha(+)-thalassemia deletion with the breakpoints in the alpha 2-globin gene and in close proximity to an Alu family repeat between the psi alpha 2- and psi alpha 1-globin genes

A novel 5.3-kb deletion of the alpha-globin gene cluster was observed in a family from Naples, Southern Italy. It removes the 5' end of the alpha 2-globin gene, causing an alpha (+)-thalassemia defect. Because of the presence of the residual 3' end of the alpha 2-globin gene, we indicated this new haplotype with the symbol (alpha)alpha 5.3. The 5' breakpoint, the first to be reported in the intergene region of the psi alpha 2- and psi alpha 1-globin genes, is located 822 bp upstream of the cap site of the psi alpha 1-gene and about 150 bp upstream of a 300- nt Alu family member. The 3' breakpoint is located in the IVS-1 nt 58 of the alpha 2-globin gene. The 5.3-kb deleted fragment shows particular characteristics: it contains four Alu sequences having long regions 80% complementary and the 5'-GGCC-3' short repeat at both ends. The sequences spanning across the breakpoints on the same strand and containing this repeat on their 3' and 5' ends, respectively, are 17 of 25 base complementary. These particular features led us to assume the formation of a multistem-loop due to the intrastrand interaction between the complementary regions as intermediate to the deletion. The unusual localization of the 5' breakpoint suggests that even the intergene region of the psi alpha 2- and psi alpha 1-globin genes may function as a deletion target.