Restriction fragment length polymorphism of the human beta-globin gene cluster: analysis of a beta-thalassemic family in Taiwan

We have analysed seven polymorphic restriction sites of the human beta-globin gene cluster of six members of a Chinese family with a beta +-thalassemic sibling. The seven polymorphic sites analysed are the HincII site at the 5'-end of the epsilon-globin gene, the HindIII sites in the two gamma-globin genes, two HincII sites within and at the 3'-end of the psi beta 1 pseudogene, the AvaII site in the beta-globin gene and the BamHI site located at the 3' side of the beta-globin gene. The beta thal chromosome has been identified to have a haplotype of +----++ with respect to these seven polymorphic sites. This is also the most predominant haplotype associated with beta +-thalassemia in Mediterranean and Chinese populations (Chen et al., 1984; Orkin et al., 1982). Of the seven sites analysed in this family, four will be useful in prenatal diagnosis of beta-thalassemia in subsequent pregnancies in the family.     

A case of prenatal diagnosis of beta-thalassemia by polymerase chain reaction]

The prenatal diagnosis of beta-thalassemia in the Udin family, where the parents were the carriers of 2 bp deletion in the codon 8 (-AA) was undertaken using PCR. Five polymorphic restriction endonuclease sites in the beta-globin gene region were tested. They are: 2 HindIII sites in the gamma G and gamma A genes, 2 HincII sites located in the pseudogene and in its 3'-flanking region, and the AvaIII site in the second exon of the beta-globin gene. The heteroduplex analysis was also performed. Two HindIII polymorphic sites were informative and the HincII site in the pseudogene and the AvaII site in the beta-globin gene were partially informative. According to the results of the RFLP analysis, the embryo was heterozygous. The similar result was obtained by heteroduplex analysis.     

A gene controlling fetal hemoglobin expression in adults is not linked to the non-alpha globin cluster.

The possible linkage between a gene causing heterocellular hereditary persistence of fetal hemoglobin (HPFH) and human non-alpha globin loci has been studied in a large Sardinian family. In this family a homozygous beta o-thalassemic patient was found, with an unusually mild form of this disease, which was ascribed to the co-existence of a gene causing heterocellular HPFH. DNA polymorphisms in the non-alpha globin cluster were analyzed by restriction enzyme digestion with HincII, HindIII and BamHI and with epsilon-, gamma-and beta-globin probes; the pattern of inheritance of these polymorphisms indicates that the HPFH gene is transmitted with one beta o-thalassemic gene in a single instance, with the second beta o-thalassemic gene in three instances and with a normal beta-globin gene in two cases. These data indicate that this HPFH gene is not linked to the non-alpha globin gene cluster, in contrast to previous observations with different HPFH genes, and suggest that this gene might code for diffusible substances acting, directly or indirectly, on gamma-globin gene expression.     

Discrimination among the human beta A, beta S, and beta C-globin genes using allele-specific oligonucleotide hybridization probes.

Synthetic nonadecanucleotides complementary to the human beta A-, beta S-, or beta C-globin sequences were used as hybridization probes to screen human genomic DNA samples for these genes. The oligonucleotides were 32P-labeled and used as probes to genotype restriction endonuclease digests of human genomic DNA. The data obtained show that hybridization with oligonucleotide probes, unlike restriction fragment length polymorphism (RFLP) analysis or direct restriction enzyme digestion, can be used to directly distinguish among the three alleles of beta-globin, beta A, beta S, and beta C, when present either in one (heterozygous) or two copies.     

Comparison of the beta-like globin gene families of rabbits and humans indicates that the gene cluster 5''-epsilon-gamma-delta-beta-3'' predates the mammalian radiation

The members of the rabbit and human beta-like globin gene families have been compared both by a computer-generated dot matrix graphical analysis of each entire gene and by calculating divergences in the coding regions. The rabbit-human gene pairs beta 4-epsilon, beta 3- gamma, psi beta 2-delta, and beta 1-beta were identified as orthologous on the basis of sequence similarities found in flanking and intervening sequences as well as by quantitative divergence calculations. The orthologous genes are in the same order on the chromosome in each species, which suggests that an ancestral family with the arrangement 5'-epsilon-gamma-delta-beta-3' preceded the mammalian radiation. Descendants of ancestral epsilon have diverged more slowly than other beta-like genes and are expressed only in embryonic life. Descendants of ancestral gamma and beta diverged at a higher rate and are expressed at wider range of developmental times. Descendants of delta have undergone nonreciprocal recombination at a high frequency and are often pseudogenes. Paralogous comparisons among the rabbit beta-like globin genes show that the beta 4-beta 3 and psi beta 2-beta 1 pairs are most similar and that beta 4 and beta 3 are more closely related to beta 1 than to psi beta 2. This fits with a branching pattern where the primordial beta split into ancestral epsilon/gamma and delta/beta genes, which later split into epsilon and gamma or delta and beta, respectively. Rabbit genes beta 4 and beta 1 acquired similar 3' untranslated regions after the epsilon/gamma split but prior to the mammalian radiation, presumably via a gene conversion event. The 5' end of beta 2 apparently converted with beta 1 after the radiation, and afterward it became a pseudogene.      

Discrimination among the transcripts of the allelic human beta-globin genes beta A, beta S and beta C using oligodeoxynucleotide hybridization probes

Three nonadecadeoxynucleotides complementary to the sense strand of the normal human beta-globin gene, beta A, and to the two allelic genes beta S and beta C were synthesized. The beta S and beta C globin genes both differ from the beta A gene by a single nucleotide substitution in the sequence coding for codon 6. The oligodeoxynucleotides are complementary to the genes in the region of the mutations and are therefore allele-specific. When radiolabeled and used as hybridization probes, the oligodeoxynucleotides are found to hybridize specifically to the mRNA transcribed from each allele.     

Analysis of the ATPase subassembly which initiates processive DNA synthesis by DNA polymerase III holoenzyme.

The gamma complex (gamma delta delta' chi psi) subassembly of DNA polymerase III holoenzyme transfers the beta subunit onto primed DNA in a reaction which requires ATP hydrolysis. Once on DNA, beta is a "sliding clamp" which tethers the polymerase to DNA for highly processive synthesis. We have examined beta and the gamma complex to identify which subunit(s) hydrolyzes ATP. We find the gamma complex is a DNA dependent ATPase. The beta subunit, which lacks ATPase activity, enhances the gamma complex ATPase when primed DNA is used as an effector. Hence, the gamma complex recognizes DNA and couples ATP hydrolysis to clamp beta onto primed DNA. Study of gamma complex subunits showed no single subunit contained significant ATPase activity. However, the heterodimers, gamma delta and gamma delta', were both DNA-dependent ATPases. Only the gamma delta ATPase was stimulated by beta and was functional in transferring the beta from solution to primed DNA. Similarity in ATPase activity of DNA polymerase III holoenzyme accessory proteins to accessory proteins of phage T4 DNA polymerase and mammalian DNA polymerase delta suggests the basic strategy of chromosome duplication has been conserved throughout evolution.     

Analysis of fibrinogen genes in patients with congenital afibrinogenemia

Several cDNA clones coding for A alpha, B beta and gamma chains of fibrinogen have been isolated from a human liver cDNA library. They were selected by differential hybridization with probes raised against fractionated liver mRNA (positive probes) and muscle and albumin mRNA (negative probes), then firmly identified by positive hybridization selection. Three of these clones, encoding A alpha, B beta and gamma fibrinogen chain sequences, were further characterized by restriction mapping and used as probes to characterize fibrinogen mRNAs from adult and fetal liver and fibrinogen genes in normal individuals and two afibrinogenemic patients. The results indicate that there is a single copy of the fibrinogen genes which are present and grossly intact in afibrinogenemic DNA.     

A multigene deletion within the immunoglobulin heavy-chain region

The immunoglobulin heavy-chain genes are located in a cluster on chromosome 14. The simultaneous absence of the human IgG1, IgG2, IgG4, and IgA1 subclasses was previously reported in a healthy Tunisian Berber and was later shown to be due to a multigene deletion. We now describe a serological and molecular study of a different deletion observed in a healthy Tunisian. Blot hybridization analysis of the proband's DNA using gamma, epsilon, alpha, and mu switch probes showed that the deletion involves a large region of the immunoglobulin heavy-chain gene cluster: C psi epsilon, C alpha 1, C psi gamma, C gamma 2, and C gamma 4. Incidentally, we showed that the restriction enzyme EcoRI alone can be used with the alpha probe to differentiate A2m types. The deletion described, present in a person homozygous for GM-Am haplotypes (Gm1,17;..;5,14,11,13,10 A2m2), is consistent with previous location, by association analysis, of C psi gamma between C alpha 1 and C gamma 2. There is evidence to suggest that deletions may be more common in the Mediterranean region than in North American Caucasians.     

A function for the psi subunit in loading the Escherichia coli DNA polymerase sliding clamp

Crystal structures of an Escherichia coli clamp loader have provided insight into the mechanism by which this molecular machine assembles ring-shaped sliding clamps onto DNA. The contributions made to the clamp loading reaction by two subunits, chi and psi, which are not present in the crystal structures, were determined by measuring the activities of three forms of the clamp loader, gamma(3)deltadelta', gamma(3)deltadelta'psi, and gamma(3)deltadelta'psichi. The psi subunit is important for stabilizing an ATP-induced conformational state with high affinity for DNA, whereas the chi subunit does not contribute directly to clamp loading in our assays lacking single-stranded DNA-binding protein. The psi subunit also increases the affinity of the clamp loader for the clamp in assays in which ATPgammaS is substituted for ATP. Interestingly, the affinity of the gamma(3)deltadelta' complex for beta is no greater in the presence than in the absence of ATPgammaS. A role for psi in stabilizing or promoting ATP- and ATPgammaS-induced conformational changes may explain why large conformational differences were not seen in gamma(3)deltadelta' structures with and without bound ATPgammaS. The beta clamp partially compensates for the activity of psi when this subunit is not present and possibly serves as a scaffold on which the clamp loader adopts the appropriate conformation for DNA binding and clamp loading. Results from our work and others suggest that the psi subunit may introduce a temporal order to the clamp loading reaction in which clamp binding precedes DNA binding.     

DNA polymorphism related to the idiopathic hemochromatosis gene: evidence in a recombinant family

Summary The metabolic error involved in idiopathic hemochromatosis, as well as the underlying genetic defect remain unknown. It has, however, been recently shown that this genetic lesion occurs at a locus linked to the major histocompatibility complex, probably close to the HLA-A locus, and that the disease is recessively transmitted. Therefore, in a family where one subject has idiopathic hemochromatosis his HLA-identical siblings should also be affected. We present here the restriction polymorphism with two MHC class I probes and one DR probe in an exceptional family with three HLA-identical siblings: one (the proband) has a major form of idiopathic hemochromatosis, while the other two are free of any clinical or biochemical signs of the disease. The restriction patterns observed after DNA digestion by enzymes EcoRI, EcoRV, BglII, BamHI, PvuII, TaqI, HincII, and HindIII led to the conclusion that one of the proband's chromosome 6 had undergone two alterations: one, a deletion in the DR region, was revealed by missing fragments all correlated with DR5; the other was an unbalanced cross-over or a genetic conversion in the MHC class I region. This latter alteration was revealed by modifications in the patterns of high molecular weight HindIII bands which hybridize with probe pHLA2 and also by the absence of a HindIII fragment of 7.4 kb hybridized by another class I probe. This latter alteration most likely involved the hemochromatosis gene and could be the first step toward a molecular approach to this gene.     

Cloning and endonuclease restriction analysis of argF and of the control region of the argECBH bipolar operon in Escherichia coli.

A 1.8 kb DNA fragment, liberated by endonuclease HindIII, contains the control region of the argECBH bipolar operon near one end and the weak secondary promoter of argH at the other extremity; it has been cloned in plasmid pBR322. The same plasmid vector has been used to clone the argF gene liberated from the chromosome by endonuclease BamHI. Restriction patterns for the two hybrid plasmids have been determined, using enzymes AluI, BglI, EcoRI, HaeIII, HincII, HindIII, HpaI and II, PstI and SalI. Two AluI sites situated on either side of and close to a HincII target delineate two short fragments covering the whole of the argECBH control region. The argF control elements are located in a region accessible to further dissection by BamHI, EcoRI, PstI and HindIII. Carriers of the argF plasmid produce extremely high amounts of ornithine carbamoyltransferase, a feature useful for purification of this enzyme.     

Versatile kanamycin-resistance cartridges for vector construction in Escherichia coli

To generate polylinker sequences which can be transferred together with an adjacent selectable marker, two plasmids (pWW-84 and pWW-97) were constructed which contain a kanamycin-resistance gene (KmR) flanked by various restriction sites. From these plasmids KmR-cartridges can be obtained as EcoRI, BamHI, SalI, AccI or HincII fragments for insertion into the appropriate restriction site of any plasmid. The following restriction sites can be introduced with these cartridges: BamHI, SalI (AccI, HincII), EcoRI, SacI, SphI and KpnI (Asp718) all adjacent to KmR, XhoI and HindIII, both within KmR. If desired, KmR can be removed by PstI digestion and religation, creating a single PstI site and leaving all adjacent sites intact.     

Molecular genetic analysis of the major histocompatibility complex in an ELA typed horse family

Restriction fragment length polymorphism was studied in an ELA typed horse family which included a stallion, a mare with two full-sibs, another mare with three full-sibs and, in addition, three paternal half-sibs. DNA samples from all individuals were investigated by Southern blot analysis using three restriction enzymes (EcoRI, HindIII or TaqI) and human cDNA class I, class II (DR beta) and class III (C4) probes. In addition, a genomic class II DQ alpha probe was used. Fragments hybridized with the various probes revealed the existence of DNA sequences homologous to HLA class I, DR beta, DQ alpha and C4 genes in the horse. Polymorphic fragments were found when DNA was hybridized with class I and class II probes irrespective of the enzyme used; but hybridization with the C4 probe did not reveal variability. All polymorphic fragments segregated according to the ELA serological specificities, thus indicating a close linkage between the different revealed subregions. Banding patterns suggest that the horse possesses about 20-30 class I genes, probably more than one DR beta and DQ alpha genes and possibly only one C4 gene. The high degree of polymorphism observed suggests that molecular DNA typing may represent a potentially powerful aid to decision in parentage control determination.     

Cloning and characterization of a gene encoding 22 kDa functional protein of bacteriophage MB78

Functional protein of MB78 bacteriophage having apparent molecular weight of 22 kDa is expressed from 1.7 kb HindIII G fragment. The nucleotide sequence of this fragment showed two open reading frames of 222 and 196 codons in tail-to-tail orientation separated by a 62-nucleotide intercistronic region. The ORF of 22 kDa protein is present in opposite orientation, i.e. in the complementary strand, preceded by a strong ribosomal binding site and a promoter sequence. Another ORF started from the beginning of the fragment whose promoter region and translational start site lies in the 0.45kb HincII U fragment which is located next to the HindIII G fragment, that has the sequence for DNA bending. 3'end of the fragment has high sequence homology to the EaA and EaI proteins of bacteriophage P22, a close relative of MB78 phage.     

Determination of β haplotypes in patients with sickle-cell anemia in the state of Rio Grande do Norte, Brazil

β(S) haplotypes were studied in 47 non-related patients with sickle-cell anemia from the state of Rio Grande do Norte, Brazil. Molecular analysis was conducted by PCR/RFLP using restriction endonucleases XmnI, HindIII, HincII and HinfI to analyze six polymorphic sites from the beta cluster. Twenty-seven patients (57.5%) were identified with genotype CAR/CAR, 9 (19.1%) CAR/BEN, 6 (12.8%) CAR/CAM, 1 (2.1%) BEN/BEN, 2 (4.3%) CAR/Atp, 1 (2.1%) BEN/Atp and 1 (2.1%) with genotype Atp/Atp. The greater frequency of Cameroon haplotypes compared to other Brazilian states suggests the existence of a peculiarity of African origin in the state of Rio Grande do Norte.     

Chromosomal assignment of genes encoding the alpha, beta, and gamma subunits of human complement protein C8: identification of a close physical linkage between the alpha and the beta loci

The eighth component of human complement (C8) is a serum protein containing three nonidentical subunits (alpha, beta, gamma) that are arranged as a disulfide-linked alpha-gamma dimer and a noncovalently associated beta chain. In earlier genetic studies, electrophoretic analysis of C8 protein polymorphisms revealed several allelic variants of alpha-gamma and beta. These were governed by separate loci designated C8A and C8B for alpha-gamma and beta, respectively. Genetic linkage analyses indicated that these loci were linked to each other and to chromosome 1 marker loci PGM1 and Rh, but it was unclear at the time if C8A was a single locus coding for a single-chain precursor form of alpha-gamma or if separate loci existed for alpha and gamma. Since evidence now indicates that alpha, beta, and gamma are encoded by separate genes, cDNA probes corresponding to each subunit were used to make direct assignments of the individual loci. Analysis of somatic cell hybrids revealed that only the alpha and beta loci are located on chromosome 1. Parallel analysis of genomic DNA digests using 5' and 3'-specific cDNA probes showed they are physically linked (less than 2.5 kb) and oriented 5' alpha-beta 3'. Further probing of the hybrid panel revealed that gamma is located on chromosome 9q. Thus, the observed genetic linkage of alpha-gamma to beta must be determined solely by alpha. In accordance with these findings, the C8 loci should now be designated C8A, C8B, and C8G for alpha, beta and gamma, respectively.     

Human globin psi B2 is not a globin-related sequence

We have determined the complete nucleotide sequence of 3.4 kilobase pairs of DNA covering the region of the human beta globin gene cluster where a human globin-related sequence psi beta 2 was thought to lie (Fritsch, Lawn, and Maniatis (1980) Cell 19, 959-972). Analysis of the resulting data reveals no evidence for any globin-related sequences in this region. The region does, however, contain several stretches of poly (dA-dT). We have confirmed the observations of Fritsch et al. that DNA from the psi beta 2 region hybridizes to the poly (dA-dT)-tailed human fetal globin cDNA plasmid, pJW151 (Wilson et al., (1978) Nucl. Acids Res. 5, 563-581) under conditions of low stringency, but we find that this hybridization is abolished by the addition of poly(rA). We conclude that psi beta 2 is not a globin pseudogene, and that the earlier investigators were probably misled by hybridization between the poly (dA-dT) stretches within the psi beta 2 region and the tails used in constructing the cDNA plasmid.     

Restriction endonuclease map of the nontoxigenic corynephage gamma c and its relationship to the toxigenic corynephage beta c.

Clear-plaque-forming mutant gamma tox- corynephages were isolated independently from nontoxigenic lysogenic Corynebacterium diphtheriae strains C7s(gamma tox-) and C4(gamma tox-). A physical map was constructed by using restriction endonucleases BamHI, EcoRI, HindIII, and KpnI. A comparison of nontoxigenic gamma c with toxigenic corynephage beta c revealed large areas of homology, including common regions for cohesive ends (cos) and attachment sites (att). Localization of the att sites on the beta prophage and correlation of the physical and genetic maps defined the orientation of the diphtheria tox operon. Diphtheria tox sequence homologies were mapped on gamma c by hybridizing 32P-labeled diphtheria tox mRNA to restriction fragments of gamma c DNA. Two regions of heterogeneity between phages beta c and gamma c were localized and these regions accounted for the 3-kilobase larger molecular size of gamma c compared with beta c. One change occurs near the tox promoter and may explain the nontoxigenic phenotype of corynephage gamma tox-.     

Comparative genetic study of group B streptococcal strains of human and bovine origin

The presence and restriction fragment length polymorphism (RFLP) of DNA fragments hybridizing with virulence and "house keeping" gene probes were analyzed for 87 group B streptococcal (GBS) strains of human and bovine origin. Most characteristics obtained for bovine strains were similar when compared with those for human strains. The most significant degree of RFLP was discovered for the sizes of HindIII fragments containing bca gene. Human GBS strains with bac gene, encoding beta antigen with IgA binding capacity, were characterized by almost identical complex hybridization patterns with multiple gene probes. At the same time bac gene was not found among bovine GBS strains. Gene scpB that encodes C5a peptidase in all human GBS strains was detected only in 9 of 39 strains of bovine origin. These two characteristics effectively distinguished bovine GBS strains from GBS strains of human origin.