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Eight classes of human leukocyte interferon (LeIFN) cDNA clones have been identified in a cDNA library prepared from a myeloblastoid cell line. The nucleotide sequences demonstrate that the multiple human LeIFN genes code for a family of homologous, yet distinct proteins. One of the cDNA clones may have been derived from the transcription of a LeIFN pseudogene.

A single recombinant lambda bacteriophage isolated from a human genome library contains two closely related human interferon genes of the leukocyte or alpha type. The two genes are separated by 12 kilobase pairs and are oriented in the same direction with respect to transcription. Comparisons of the DNA sequences of these two genes and interferon complementary DNA clones indicate that the two interferon genes lack intervening sequences.

Lipoprotein lipase is a key enzyme of lipid metabolism that acts to hydrolyze triglycerides, providing free fatty acids for cells and affecting the maturation of circulating lipoproteins. It has been proposed that the enzyme plays a role in the development of obesity and atherosclerosis. The human enzyme has been difficult to purify and its protein sequence was heretofore undetermined. A complementary DNA for human lipoprotein lipase that codes for a mature protein of 448 amino acids has now been cloned and sequenced. Analysis of the sequence indicates that human lipoprotein lipase, hepatic lipase, and pancreatic lipase are members of a gene family. Two distinct species of lipoprotein lipase messenger RNA that arise from alternative sites of 3'-terminal polyadenylation were detected in several different tissues.

Four hybrid human leukocyte interferon (IFN-alpha) genes have been constructed and expressed in Escherichia coli using molecular cloning methods. Plasmids containing genes encoding human interferons, IFN-alpha A, IFN-alpha D, IFN-alpha I and several hybrids of the different IFN-alpha genes (formed by in vitro recombination at common restriction endonuclease sites located within the DNA sequence encoding mature polypeptides) joined identically to an E. coli trp promoter gave rise to bacterial-produced interferons with distinctly different antiviral activities. The expression plasmid which directs the synthesis of IFN-alpha I was constructed using a gene isolated from a human genomic library in a manner similar to the previous expression of IFN-alpha A and IFN-alpha D cDNA clones. The use of a cell-free transcription-translation system has allowed the calculation of the specific activities of IFN-alpha made from isolated DNA fragments containing these hybrid IFN-alpha genes. These bacterially-derived interferons vary considerably in their ability to inhibit vesicular stomatitis virus (VSV) in different mammalian cells. The results show that the cloned hybrid interferons have unique antiviral activities when compared with the parent interferons, and they demonstrate that more active IFN-alpha s can be made using recombinant DNA techniques.

A cloned library of large, random embryonic human DNA fragments was constructed and screened for beta-globin sequences using the cloned human beta-globin cDNA plasmid pJW102 (Wilson et al., 1978) as a hybridization probe. Two independent clones were obtained and then characterized by restriction endonuclease cleavage analysis, hybridization experiments and partial DNA sequencing. Each of the clones carries both the adult delta- and beta-globin genes. The two genes are separated by approximately 5.4 kilobases (kb) of DNA and their orientation with respect to the direction of transcription is 5'-delta--beta-3'. Both the delta- and beta-globin genes contain a large noncoding intervening sequence (950 and 900 bp, respectively) located between the codons for amino acids 104 (arginine) and 105 (leucine). Although the location of the large intervening sequence within the coding regions of the two genes is identical, the two noncoding sequences bear little sequence homology. A second, smaller intervening sequence similar to that found in other mammalian beta-globin genes was detected near the 5' end of the human beta-globin gene. The two independently isolated beta-globin clones differ from each other by the presence of a Pst I restriction enzyme cleavage site within the large intervening sequence of the delta-globin gene of one of the clones. This suggests that the human DNA carried in the two clones was derived from two homologous chromosomes which were heterozygous for the Pst I restriction enzyme recognition sequence.

A recombinant plasmid has been constructed which contains the mature protein coding region of the human serum albumin (HSA) gene. Bacteria containing this plasmid synthesize HSA protein under control of the E. coli trp promoter-operator. The DNA sequence and predicted protein sequence of HSA were determined from the cDNA plasmid and are compared to existing data obtained from direct protein sequencing. The DNA sequence predicts a mature protein of 585 amino acids preceded by a 24 amino acid "prepro" peptide.

The transfer of insoluble cholesteryl esters among lipoprotein particles is a vital step in normal cholesterol homeostasis and may be involved in the development of atherosclerosis. Extrahepatic tissues lack the enzymes required for the degradation of sterols to the excretable form of bile acids. Cholesterol synthesized in these tissues in excess of that needed for the synthesis of cell membranes or steroid hormones must accordingly be returned through the plasma to the liver for catabolism. The series of reactions involved has been termed reverse cholesterol transport. Catalysed steps of this pathway are believed to include an efflux from peripheral cells, which generates a diffusion gradient between these membranes and extracellular fluid; esterification of this cholesterol by lecithin-cholesterol acyltransferase (LCAT) (phosphatidylcholine-sterol acyltransferase) acting on species of high-density lipoproteins; transfer of the cholesteryl esters formed (largely to low- and very low-density lipoproteins) (LDL and VLDL) by a cholesteryl ester transfer protein (CETP); and removal of these lipoproteins, together with their cholesteryl ester content, by the liver through receptor-mediated and nonspecific endocytosis. Of these steps, the CETP reaction is the least characterized. Several laboratories have reported the purification from human plasma of proteins active on cholesteryl ester transfer between lipoprotein particles and possibly between cells and plasma. However, the reported relative molecular mass (Mr), abundance and specificity of the purified activities have differed considerably. We have recently described the preparation of a highly active CETP of Mr 74,000 purified about 100,000-fold from human plasma, which may represent the functional component of earlier preparations. Using a partial amino-acid sequence from this purified protein, CETP complementary DNA derived from human liver DNA has been cloned and sequenced and the cloned DNA used to detect CETP messenger RNA in a number of human tissues.

At least ten leukocyte interferon genes and the single known fibroblast interferon gene have been localized on the pter leads to q12 region of human chromosome 9. Gene mapping was accomplished by blot hybridization of cloned interferon complementary DNA to DNA from human-mouse cell hybrids with a translocation involving human chromosome 9. Supporting evidence suggests these genes are clustered.

Recombinant plasmids containing cDNA inserts from human leukocyte interferon (IFN-alpha), fibroblast interferon (IFN-beta), or immune interferon (INF-gamma) genes were radiolabeled and hybridized in situ to human metaphase chromosome preparations. The results localized the human IFN-alpha and IFN-beta genes to the short arm of chromosome 9(p21-->pter) and localized the IFN-gamma gene to the long arm of chromosome 12(q24.1).

We present the results of a detailed comparison of the primary structure of human beta-like globin genes and their flanking sequences. Among the sequences located 5' to these genes are two highly conserved regions which include the sequences ATA and CCAAT located 31 +/- 1 and 77 +/- 10 bp, respectively, 5' to the mRNA capping site. Similar sequences are found in the corresponding locations in most other eucaryotic structural genes. Calculation of the divergence times of individual beta-like globin gene pairs provides the first description of the evolutionary relationships within a gene family based entirely on direct nucleotide sequence comparisons. In addition, the evolutionary relationship of the embryonic epsilon-globin gene to the other human beta-like globin genes is defined for the first time. Finally, we describe a model for the involvement of short direct repeat sequences in the generation of deletions in the noncoding and coding regions of beta-like globin genes during evolution.

A partial cDNA clone for human antithrombin III (ATIII) was obtained by screening a cDNA library prepared from size fractionated liver RNA with a pool of eight 16-base long synthetic DNA fragments whose sequence was determined from protein sequence data. A fragment of the partial cDNA clone was used to enrich RNA for ATIII messages, and cDNA clones encoding the entire ATIII structural gene were identified. The complete nucleotide and predicted amino acid sequences of human ATIII and its 32 residue signal peptide are reported, and provide further opportunity to compare the ATIII primary structure with corresponding regions from homologous proteins, alpha 1-antitrypsin and ovalbumin. Plasmids in which the structural genes for mature and pre-ATIII were linked to the E. coli trp promoter-operator support the synthesis of human antithrombin III and pre-antithrombin III in bacteria.

Tissue factor is a membrane protein that plays an essential role in the initiation of blood coagulation. When exposed to the circulation, tissue factor interacts with the serine protease factor VII, and the complex triggers fibrin clot formation by activating both factors IX and X of the coagulation cascade. This report describes the cloning and expression of the complementary DNA (cDNA) for human tissue factor. The cDNA encodes a protein of 263 amino acids preceded by a 32 amino acid signal peptide. The predicted protein sequence contains a potential hydrophobic membrane anchoring domain at its carboxy terminus, and bears no significant homology to any other known protein. Tissue factor mRNA of 2400 nucleotides was detected in adipose, adrenal, small intestine and a number of other tissues by Northern blot hybridization analysis. In order to confirm the identity of the cDNA, an expression vector containing the cloned cDNA was used to transfect cultured mammalian cells. These cells produced active tissue factor which was assayed using purified factors VII and X.

The human lecithin-cholesterol acyltransferase (LCAT) gene has been sequenced to completion. The gene is divided into six exons spanning approximately 4,200 bp. Exon five codes for amino acids homologous to the interfacial active site of several lipases, and also codes for an amphipathic alpha-helix resembling the carboxy terminus of apolipoprotein E. Blot hybridization data suggest that there is only one LCAT gene in humans. The 1550 base LCAT mRNA can be detected in liver and HepG2 (hepatocyte) cells, but not in small intestine, spleen, pancreas, placenta or adrenal tissue.

The gene for human leukocyte interferon alpha 2 (designated either LeIF A or HuIFN-alpha 2) has been isolated from a human genome library. The DNA sequence of this gene demonstrates that it lacks introns. The 3' noncoding sequences of the IFN-alpha 2 gene correspond to two types of IFN-alpha 2 cDNA clones we have isolated that have alternate sites of polyadenylylation. A comparison of seven human IFN-alpha sequences shows that they are homologous in the 5' flanking region and contain identical "TATA box" sequences. The recombinant lambda clone containing the IFN-alpha 2 gene also contains two copies of the "Alu family" repeat sequence.

At least eight leukocyte interferon genes (IFL) and the single fibroblast interferon gene (IFF) have been located on chromosome 9 in humans. In somatic cell hybrids of human and mouse cells containing a normal complement of mouse parental cell chromosomes but reduced numbers of human chromosomes, the human leukocyte and fibroblast interferon DNA sequences were present only when human chromosome 9 was also present.

DNA clones encoding the complete 2,351 amino acid sequence for human factor VIII have been isolated and used to produce biologically active factor VIII in cultured mammalian cells. The recombinant protein corrects the clotting time of plasma from haemophiliacs and has many of the biochemical and immunological characteristics of serum-derived factor VIII.

Apolipoprotein(a) [apo(a)], the glycoprotein associated with the lipoprotein(a) [Lp(a)] subfraction of plasma lipoproteins, has been shown to exhibit heritable molecular weight isoforms ranging from 400-700 kDa. Increased serum concentrations of Lp(a) correlate positively with the risk of atherosclerosis. Variations in Lp(a) plasma levels among individuals are inherited as a codominant quantitative trait. As part of an effect to define the basis of these variations and further clarify the expression of the protein, we have determined the chromosomal location of the human apo(a) gene. Blot hybridization analysis of DNA from a panel of mouse-human somatic cell hybrids with an apo(a) cDNA probe revealed a complex pattern of bands, all of which segregated with chromosome 6. In situ hybridization yielded a single peak of grain density located on chromosome 6q26-27. Apo(a) cDNA sequences exhibit striking homology to those of the plasma protease plasminogen, and, therefore, we have reexamined the chromosome assignment of the plasminogen gene. We conclude that both the apo(a) and plasminogen genes reside on human chromosome 6q22-27, consistent with a gene duplication mechanism for their evolutionary origin. The results are of significance for the genetic control of apo(a) expression and genetic influences predisposing to atherosclerosis.

cDNA and genomic cloning has been used to determine the mRNA and amino acid sequence of human plasma lecithin-cholesterol acyltransferase (LCATase; EC 2.3.1.43). The mature protein was found to contain 416 amino acid residues with a hydrophobic leader sequence of 24 amino acids. An unusual feature of the message is that the poly(A) signal AATAAA overlaps the COOH-terminal glutamic acid and stop codons, and the 3' untranslated region is only 23 bases. The protein itself is distinguished by a number of extended sequences of hydrophobic amino acids, one of which contains a hexapeptide identical with the interfacial binding segment of the active site of pancreatic lipase and is similar to the same site of lingual lipase. The cloned cDNA allows the expression of active LCATase by transfected tissue culture cells.

The exons and bordering intron nucleotides of the human apolipoprotein D (apo D) gene have been sequenced. The protein-coding portion of the gene is divided into five exons which span approximately 12,000 bp. At least one intron interrupts the 5' untranslated region. The gene has been localized to the p14.2----qter region of human chromosome 3. Apo D shares homology with the alpha 2u-globulin superfamily of genes, including approximately 25% amino acid homology with human retinol-binding protein (RBP). Similarity of intron locations in both apo D and RBP suggests that these two genes derived from a common ancestor.

Human lipoprotein(a) is a low density lipoprotein-like lipoprotein whose concentration in plasma is correlated with atherosclerosis. The characteristic protein component of lipoprotein(a) is apolipoprotein(a) (apo(a)) which is disulfide-linked to apolipoprotein B-100. Sequencing of rhesus monkey apo(a) cDNA suggests that this protein, like human apo(a), is highly similar to plasminogen. Sequence data suggests that a plasminogen-like protease activity and kringle 1-, 2-, 3-, and 5-like domains are unnecessary for apo(a) function, but a highly repeated kringle four-like domain is important. Liver is the major site of apo(a) RNA synthesis; reduced amounts of message were also found in testes and brain. Co-expression with apoB-100 and plasminogen in rhesus tissues is not mandatory.

Apolipoprotein(a) [apo(a)] is a glycoprotein with Mr approximately equal to 280,000 that is disulfide linked to apolipoprotein B in lipoprotein(a) particles. Elevated plasma levels of lipoprotein(a) are correlated with atherosclerosis. Partial amino acid sequence of apo(a) shows that it has striking homology to plasminogen. Plasminogen is a plasma serine protease zymogen that consists of five homologous and tandemly repeated domains called kringles and a trypsin-like protease domain. The amino-terminal sequence obtained for apo(a) is homologous to the beginning of kringle 4 but not the amino terminus of plasminogen. Apo(a) was subjected to limited proteolysis by trypsin or V8 protease, and fragments generated were isolated and sequenced. Sequences obtained from several of these fragments are highly (77-100%) homologous to plasminogen residues 391-421, which reside within kringle 4. Analysis of these internal apo(a) sequences revealed that apo(a) may contain at least two kringle 4-like domains. A sequence obtained from another tryptic fragment also shows homology to the end of kringle 4 and the beginning of kringle 5. Sequence data obtained from two tryptic fragments show homology with the protease domain of plasminogen. One of these sequences is homologous to the sequences surrounding the activation site of plasminogen. Plasminogen is activated by the cleavage of a specific arginine residue by urokinase and tissue plasminogen activator; however, the corresponding site in apo(a) is a serine that would not be cleaved by tissue plasminogen activator or urokinase. Using a plasmin-specific assay, no proteolytic activity could be demonstrated for lipoprotein(a) particles. These results suggest that apo(a) contains kringle-like domains and an inactive protease domain.

The deduced amino acid sequence of human factor VIII, obtained from the DNA sequence, predicts a mature polypeptide of 2,332 amino acids containing a triplicated domain structure. The polypeptide has 35% sequence homology with the copper-binding plasma protein, ceruloplasmin. Determination of the thrombin cleavage sites in plasma-derived factor VIII polypeptides allows prediction of the domains involved in the associated activation and inactivation of the protein.

Individuals heterozygous for the Greek (A gamma) variant of hereditary persistence of fetal haemoglobin (HPFH) synthesize Hb F whose gamma-globin chains are predominantly of the A gamma type. DNA obtained from Greek HPFH heterozygotes was used to test for abnormalities in the organization of non alpha-globin genes. In addition, gamma- and beta-globin expression was studied in BFUe cultures. Restriction endonuclease mapping showed that the G gamma, delta and beta genes in cis to the Greek HPFH determinant are intact. Overproduction of gamma-globin chains synthesis was observed in the BFUe cultures. A significant portion of the gamma chain synthesis was of the G gamma type, suggesting that the G gamma genes cis and trans to the HPFH chromosome are active in culture. DNA mapping data indicate that in contrast to G gamma A gamma HPFH and the G gamma (delta beta) thalassaemia, the Greek (A gamma) HPFH is not due to a large deletion in the non-alpha globin gene region. It is possible that the anomaly may result either from a small deletion or point mutation which influences non alpha-globin transcription. The in vitro synthesis data suggest that the low level of G gamma-globin chain synthesis in vivo is not the result of transcriptional inactivation of the G gamma gene, since this gene appears to be expressed in erythroid cell cultures. We speculate that the genetic lesion in Greek (A gamma) HPFH is in regulatory sequences which control the level of G gamma and A gamma expression during development.

Cholesteryl ester transfer protein (CETP) may have important roles in transfer of lipids from cells to serum lipoproteins or between circulating lipoprotein particles. Restriction fragment length polymorphisms (RFLPs) in DNA at the CETP locus have been detected. In the present study we have used RFLPs detectable with the restriction enzyme TaqI to examine if CETP influences serum lipid variability (as opposed to absolute lipid levels). We have compared within-pair difference in serum lipid and apolipoprotein levels in monozygotic twin pairs of various genotypes in the B polymorphism at the CETP locus and uncovered significant differences between genotypes. We conclude that the CETP locus has "variability genes" (as opposed to "level genes") with respect to total and LDL cholesterol variability. A person's total genetic risk for coronary heart disease may depend on his or her combination of "level genes" and "variability genes". The method of analysis applied may be the best available for the study of gene - environment interaction.

We have used a cDNA probe for human cholesteryl ester transfer protein (CETP) to determine the chromosomal location for the human gene. Southern blot analysis of DNA from 17 independent mouse-human somatic cell hybrids demonstrated the presence of the gene for human CETP on chromosome 16. Regional mapping of the gene by in situ hybridization was consistent with these results and indicated that the gene resides in the 16q12-21 region of the chromosome. These findings provide an additional polymorphic marker for chromosome 16, as several relatively common restriction fragment length polymorphisms of the gene have previously been reported, and they have significance for studies directed at the identification of genetic factors affecting plasma lipoprotein metabolism and atherosclerosis.

The primary structure of factor VIII consists of 2332 amino acids that exhibit 3 distinct structural domains, including a triplicated region (A domains), a unique region of 909 amino acids (B domain), and a carboxy-terminal duplicated region (C domains), that are arranged in the order A1-A2-B-A3-C1-C2. The B domain (residues 741-1648) of factor VIII is lost when factor VIII is activated by thrombin, which proteolytically processes factor VIII to active subunits of Mr 50,000 (domain A1), 43,000 (domain A2), and 73,000 (domains A3-C1-C2). To determine if the B domain is required for factor VIII coagulant activity, a variant was constructed by using recombinant DNA techniques in which residues 797-1562 were eliminated. This shortened the B domain from 909 to 142 amino acids. This variant factor VIIIdes-797-1652 was expressed in mammalian cells and was found to be functional. The factor VIIIdes-797-1562 protein was purified and shown to be processed by thrombin in the same manner as full-length factor VIII. The factor VIIIdes-797-1562 variant also bound to von Willebrand factor (vWF) immobilized on Sepharose. These results indicate that most of the highly glycosylated B domain of factor VIII is not required for the expression of factor VIII coagulant activity and its interaction with vWF.

Cholesteryl ester transfer protein (CETP) is a protein involved in "reverse cholesterol transport" and it could play an important role in facilitating the removal of cholesteryl esters from peripheral tissues for transport to the liver or for transfer of cholesterol between plasma lipoprotein particles. Both functions may be relevant to susceptibility or resistance to atherosclerotic disease. We have studied 149 and 146 unrelated persons, respectively, for the A and B polymorphism at the CETP locus detectable with the restriction enzyme TaqI. The B system is by far the more polymorphic. A search for association with risk or "anti-risk" factor levels was conducted with the following quantitative parameters: total cholesterol, HDL cholesterol, triglycerides, apolipoprotein AI (apoA-I), apolipoprotein B (apoB) and Lp(a) lipoprotein levels. Highly significant differences in apoA-I concentration were found between the two categories of homozygotes in the B polymorphism. The association observed remained significant after multiplying the p value by the number of quantitative parameters used for the association tests. There was a dosage effect on the apoA-I level of genes in the B polymorphism. We conclude that the associations observed are likely to reflect true biological phenomena. The effect of CETP genes appeared to be limited to non-smokers.

An oligonucleotide hybridization procedure has been developed that eliminates the preferential melting of A X T versus G X C base pairs, allowing the stringency of the hybridization to be controlled as a function of probe length only. This technique, which uses tetramethylammonium chloride, is especially helpful whenever a highly complex library is screened with a pool of oligonucleotide probes, which usually vary widely in base composition. The procedure can also be applied advantageously whenever an exact match to an oligonucleotide probe is desired, such as in screening for clones having as little as a single-base alteration generated by in vitro mutagenesis.

The most common inherited bleeding disorder in man, haemophilia A, is caused by defect in factor VIII, a component in the blood coagulation pathway. The X-chromosome-linked disease almost certainly stems from a heterogeneous collection of genetic lesions. Because, without proper treatment, haemophilia can be a fatal disease, new mutations are necessary to account for its constant frequency in the population. In addition, haemophilia A displays a wide range of severity, and some 15% of haemophiliacs generate high levels of antibodies against factor VIII ('inhibitor patients'). The present work elucidates the molecular genetic basis of haemophilia in some individuals. Using the recently cloned factor VIII gene as a probe, we have identified two different nonsense point mutations in the factor VIII gene of haemophiliacs, as well as two different partial deletions of the gene. Our survey of 92 haemophiliacs indicates no firm correlation between antibody (inhibitor) production and gross gene defects.

Deletions in the DNA of individuals with hereditary persistence of fetal haemoglobin (HPFH) and 8 beta-thalassaemia have been mapped as a means of identifying regulatory sequences involved in the switch from fetal to adult globin gene expression. The end points of these deletions have been precisely located with respect to restriction endonuclease cleavage sites within and surrounding the gamma-, delta- and beta-globin genes in normal human DNA and the deletion maps were used to obtain definitive evidence for the physical linkage of the fetal and adult beta-like globin genes in the order 5'Ggamma-Agamma-delta-beta 3'. Correlation of haematological data and the location of deletions in two cases of HPFH and one case of deltabeta-thalassaemia suggest that a region of DNA located near the 5'-end of the delta-globin gene may be involved in the suppression in cis of gamma-globin gene expression in adults. The interpretation of a second case of deltabeta-thalassaemia is complicated by the fact that the deletion removes the Agamma-gene in addition to the region near the 5'-end of the delta-globin gene.

Lipoprotein(a) is an LDL-like lipoprotein whose concentration in plasma is correlated with atherosclerosis. The characteristic protein component of lipoprotein(a) is apolipoprotein(a) which is disulphide-linked to apolipoprotein B-100. Sequencing of cloned human apolipoprotein(a) complementary DNA shows that it is very similar to human plasminogen. It contains a serine protease domain and two types of plasminogen-like kringle domains, one of which is present in 37 copies.

The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.

A recombinant lambda bacteriophage isolated from a human genome library contains the gene for fibroblast interferon (IFN-beta1). The DNA sequence of this gene is identical to the sequence of its mRNA and is devoid of introns.

DNA probes derived from the cloned factor VIII gene can be used to detect mutations in the factor VIII gene of hemophiliacs. DNA hybridization analysis led to the identification of two contrasting point mutations in the same codon. In a severe hemophiliac with no detectable factor VIII activity, the normal arginine codon (number 2307) is converted to a stop codon, while in a mild hemophiliac with 10 percent of normal activity, this same codon is converted to glutamine.

Haemophilia A is the most common inherited bleeding disorder in man, affecting approximately 1 male in 10,000. The disease is caused by a deficiency in the gene for factor VIII, a component of the intrinsic coagulation pathway. Due to the broad range of clotting activity in normal and heterozygous females, it is often difficult to confirm the status of women at risk for carrying the disease. A genetic marker in the form of a restriction fragment length polymorphism (RFLP) within or tightly linked to the factor VIII gene would serve as a tag for the haemophilia gene, thus allowing both accurate carrier detection and improved, earlier prenatal diagnosis by chorionic villi sampling. The recent isolation of the factor VIII gene has allowed a search for RFLPs within the gene, and we report here the identification of a common polymorphism within the factor VIII gene, revealed by the restriction enzyme BclI, which can be used diagnostically in about 42% of all families. Although the disease haemophilia A has been mapped to the distal portion of Xq, the BclI RFLP makes possible higher-resolution genetic linkage mapping with respect to other polymorphic markers on this portion of the X chromosome. We have established close linkage of the factor VIII gene to several useful RFLP markers, including the highly informative marker St14. These markers should also be useful for prenatal diagnosis of haemophilia A and for detection of its carriers.

A DNA polymorphism for an Xbal site in intron 22 of the human factor VIII:C gene extends the utility of DNA methods for carrier detection in families segregating for hemophilia A. While the DNA polymorphism detected by a BclI site in intron 18 of the factor VIII:C gene was informative for 41% of females studied, the BglI/intron 25 polymorphism provided no additional information because of apparent linkage disequilibrium. In contrast, the Xbal intron 22 polymorphism was useful in 53% of women who were uninformative (homozygous) for either the BclI or BglI polymorphisms. Using the BclI/intron 18 and Xbal/intron 22 intragenic polymorphisms, we could provide highly accurate information for 68% of women we studied who were at risk for carriership. The carrier status of the remaining 32% could be determined utilizing the closely linked Taql/St14 DNA polymorphism.

The cellular site of synthesis of factor VIII (FVIII:C; anti-haemophilic factor) has long been sought. Previous studies suggested the liver as a major site of synthesis, but extrahepatic sources such as spleen and lung have been implicated. Using an immunoradiometric assay (IRMA), we recently localized factor VIII antigen (FVIII:Ag, formerly FVIII:CAg), to whole perfused guinea pig liver and spleen, and to isolated hepatocytes, with lesser or trace amounts in other tissues. Using an immunohistological technique, Stel et al. detected FVIII:Ag in normal human liver sinusoidal endothelial cells, while Exner et al. detected FVIII:Ag by IRMA in extracts of human lymph nodes, lung, liver and spleen. The localization of antigen in tissues does not, however, distinguish sites of factor VIII synthesis from those of storage, and such experiments are subject to misinterpretation due to entrapment of plasma factor VIII in tissues. The recent cloning of the human factor VIII gene provides hybridization probes for the detection of factor VIII messenger RNA in cells, thus directly determining sites of synthesis. During complementary DNA cloning, we detected factor VIII mRNA in liver, and it has been localized by others in liver and placenta and in liver and kidney. In the present study, we detected factor VIII mRNA in isolated human hepatocytes, in spleen and in numerous tissues including lymph nodes and kidney, but not in white blood cells or cultured endothelial cells. We also found that the factor VIII, factor VII, factor IX and protein C antigens in liver are predominantly localized in hepatocytes, while very little von Willebrand factor antigen (vWF:Ag, formerly FVIIIR Ag) is detectable in this organ.