Alterations In Gene Structure Research Articles

Molecular Basis for the Human Erythrocyte Glycophorin Specifying the Miltenberger Class I (Mil) Phenotype

Abstract Human glycophorin Mil (HGpMil) is a structural variant of the MNSs blood group system that specifies the Miltenberger class I phenotype. We report here the molecular basis of the HGpMil gene identified in a white family in which the first homozygote was encountered. Immunoblotting analysis showed the expression of HGpMil and HGpB but the absence of HGpA on the homozygous Mil erythrocytes. Southern blot analysis detected no gross alterations in gene structure or band intensity. Genomic sequences encompassing exons II and III of the HGpMil gene were amplified by single-copy polymerase chain reaction. Restriction digestion and direct DNA sequence analysis showed that HGpMil gene is derived from an alpha N allele of HGpA and differs from the latter in the third exon by a single nucleotide change. In HGpMil, the presence of a deoxythymidine at the second position of codon 28 (ATG) not only resulted in a methionine substitution but also altered the consensus sequence for N-glycosylation from Asn-Asp-Thr to Asn-Asp- Met. These data are consistent with the occurrence of Mil on the red blood cell membrane as a variant deficient in the asparagine-linked carbohydrate unit. Significantly, this particular point mutation lies in between the two half-sites of a direct repeat that has been implicated to facilitate the recombination events leading to several other glycophorin genes of the Miltenberger series. Based on this relatedness, we propose an untemplated nucleotide replacement resulting from a gene conversion event as the molecular basis for the origin of HGpMil gene.

The mouse S‐antigen gene

We have characterized a gene for mouse S-antigen and compared its sequence with that of corresponding human and two recently published Drosophila S-antigen genes. The mouse S-antigen gene was approximately 50 kbp in length and consisted of 16 exons and 15 introns. The length of most exons was less than 100 bp and the smallest one was only 10 bp. In contrast, the length of most introns was larger than 2 kbp and the gene consisted of 97% intron and 3% exon. Both splice sites for donor and accepter were in good agreement with the GT/AG rule. S-antigen genes in human and mouse were highly conserved. In contrast, genes for the Drosophila 49-kDa arrestin homolog and arrestin consist of three introns and four exons and two introns and three exons, respectively. The 5'-flanking region of the mouse S-antigen gene, approximately 1.0 kbp long, had no regulatory elements for transcription such as the TATA, CAAT and GC boxes, while a Drosophila arrestin gene has TATA and CAAT boxes. Interestingly, the 5'-flanking region of the mouse gene had promoter activity in an in vitro transcription assay using a nuclear extract of rat brain. A major transcription start site was found at 387 bp upstream from the translation start codon ATG in mouse. From our results, and those of others, we suggest that the S-antigen gene has evolved from a common ancestor gene by either insertion or deletion of introns. Such an alteration of gene structure may have played a role in the evolution of the S-antigen.

Genetic heterogeneity of propionic acidemia: Analysis of 15 Japanese patients

Propionic acidemia is an autosomal recessive metabolic disease resulting from a deficiency of propionyl CoA carboxylase (PCC) activity. We have analyzed the molecular heterogeneity of Japanese propionic acidemia patients using anti-human PCC antiserum and cDNA clones coding for the two protein subunits (alpha and beta) of the enzyme. The steady state levels of both alpha and beta subunits of PCC from 15 Japanese patients were determined by Western blot. Three patients had neither alpha nor beta subunits, and the amounts of both alpha and beta subunits were low in 3 other patients. According to our previous data, we classified these 6 patients as having alpha subunit deficiency. In the remaining 8 patients, alpha subunits were normal, but the beta subunits were aberrant. Two patients had low levels of normal-sized beta subunits and 6 had beta subunits smaller than normal in size and greatly reduced in quantity. These 8 patients were assigned to the beta subunit deficiency category. One patient had apparently normal alpha and beta subunits. We could not determine this patient's primary defect. These data reveal the genetic heterogeneity of molecular defects causing propionic acidemia in the Japanese. Southern blot analysis did not reveal any gross alteration in gene structure when DNA was digested with HindIII, EcoRI and TaqI. However, DNA from 3 beta-subunit-deficient patients, when digested with MspI and probed with beta PCC cDNA, revealed a unique 2.7-kb band not observed in blots of DNA from any other patient or 15 normal controls. We conclude that this altered MspI restriction map is the result of a mutation in the beta subunit gene of these patients.

Cytosine arabinoside enhancement of gamma irradiation induced mutations in human t-lymphocytes

The frequency of 6-thioguanine resistant (TGr) mutants induced in human G0 phase T-lymphocytes by 200 cGy of gamma irradiation is greatly enhanced by incubation with cytosine arabinoside (ara-C) after irradiation. The mutant frequency increased with increasing incubation time in ara-C for up to 2 hr. This mutation induction required a phenotypic expression time of 5-8 days mass culture growth, similar to that found with mutants induced by 300 cGy of irradiation alone. Southern blot analysis of 40 isolated mutant clones revealed 8 independent mutations by T-cell receptor (TCR) gene rearrangement patterns. Four of these eight showed hprt gene structural alterations (0.50). An alternative method to allow phenotypic expression was developed to minimize the isolation of hprt/TCR sibling mutants. The use of in situ expression in the microtiter dish wells resulted in the isolation of 17 independent mutations in 19 mutant clones. Ten of these 17 mutations showed hprt structural alterations (0.59). The high fraction of mutations involving structural alterations detected by Southern blot analysis is consistent with the known induction of chromosome aberrations by irradiation plus ara-C treatment. We propose that both the increase in Mf and the increase in the incidence of hprt gene structural alterations are due to the accumulation of strand breaks in repairing regions of DNA under these conditions of ara-C induced inhibition of repair. We further propose that upon release of the ara-C inhibition, these repairing regions can interact to yield both gene mutations and chromosome aberrations.

Review of the molecular characteristics of gene mutations of the germline and somatic cells of the human

Molecular analyses of the limited number of de novo germinal mutations identified in humans indicate that an array of alterations in gene structure can be generated. Similar conclusions are derived from the large data set obtained from molecular analyses of alleles that segregate in the human population and cause genetic diseases. The molecular alterations include nucleotide substitutions as well as insertions, deletions and other rearrangemets of the DNA. The lesions may be located in the coding or the noncoding regions of genes or may involve the flanking sequences. The insertions and deletions involve fragments ranging from single nucleotides to many kilobases, and involve both unique sequences and repetive elements. The nature of the lesions observed to date as either de novo mutations or segregating variants suggests there are locus-specific characteristics of the alterations in DNA structure that are recovered as genetic diseases. Differences in mutation spectra among genetic loci appear to reflect both the structure of the target sequences and the relationship between gene structure and gene function. No induced germinal mutations have been identified, thus no data are available that reveal the relationships between mutagenic exposures and the molecular fingerprints of the lesion induced in the human germ cell and transmitted to the subsequent generations. In contrast, the prospects for analyzinng the roles of genetic target, exposure history and individual responsiveness to exposure in creating particular molecular lesions in somatic cells are excellent, both for alterations of single nucleotides and for major alterations of gene structure. By analysis of mutations at specific loci, it is possible to optimize recovery and subsequent detailed analysis of particular types of molecular events. The number of loci currently under study is small, but as more data are accumulated on the molecular nature of somatic mutation and exposure history, and more genetic targets come under study, it may be possible to identify molecular fingerprints of mutagen exposure. Somatic cells then will not only provide insight into the relationship between exposure to mutagens and somatic events such as cancer, but also serve as surrogate dosimeters for induction of heritable mutation.

Molecular analyses of in vivo hprt mutations in human T-lymphocytes III. Longitudinal study of hprt gene structural alterations and T-cell clonal origins

The hprt clonal assay detects mutations occurring in vivo in the hypoxanthine-guanine phosphpribosyltransferase ( hprt) gene of human T-lymphocytes. Analysis of 94 wild-type and 326 hprt mutant clones from 3 normal males was performed using Southern blotting with hprt and T-cell receptor (TCR) gene probes. Gross structural alterations of the hprt gene occurred in ∼ 14% of the in vivo derived mutants. Breakpoints were randomly distributed across the gene with one possible mutational “hot spot” observed. Most hprt mutants were independent as judge by TCR gene rearrangement patterns indicating that the measured hprt mutant frequency is a good measure of the actual hprt mutation frequency. However, sibling mutants (generally doublets and triplets except for one nonamer) were detected. Information on the timing in vivo of the hprt mutational events and the persistence in vivo of sibling mutants was also obtained.

Increased expression of the epidermal growth factor receptor gene in malignant gliomas is invariably associated with gene amplification.

Primary malignant gliomas from 63 patients were analyzed to determine the relationship between amplification of the gene encoding the epidermal growth factor receptor (EGFR) and expression of the corresponding mRNA. Twenty-four tumors were found to have amplified the EGFR gene and amplification of other genes occurred in three additional tumors. Hybridization with synthetic RNA probes was used to quantitate mRNA levels in situ. All 24 tumors with amplification of the EGFR gene had high levels of expression of this gene, while none of the 39 tumors without amplification had increased levels. This shows that, in human gliomas, large increases in the expression of the EGFR gene are invariably associated with alterations in gene structure.

Molecular-genetic analysis of myc and c-Ha-ras proto-oncogene alterations in human carcinoma.

The results of recent studies on the molecular biology of cancer suggest the abnormal expression of cellular proto-oncogenes in the process of carcinogenesis [1]. The activation of proto-oncogenes, i.e., their conversion into cellular oncogenes, is associated with gene structural alterations and mutations resulting in the unregulated production of on-coproteins, matched by deranged biological function of the latter. Recently, “mixed” variants of ras-family gene activation were established. They involve both amplification and point mutations in one of the “hot” codons [2, 3].

Structure and sequence of mutations induced by ionizing radiation at selectable loci in Chinese hamster ovary cells

The spectrum of mutations induced by ionizing radiation at two non-essential genetic loci varies markedly. Those at the adenine phosphoribosyl transferase ( aprt) locus predominantly have no detectable alterations of gene structure on Southern blots, while those at the hypoxanthine guanine phosphoribosyl transferase ( hprt) locus are largely massive deletions eliminating all coding sequence. Insertion mutations were detected at both loci. To characterize the sequence alterations producing the minor changes at the ( aprt) locus, two mutant genes were cloned from λ genomic libraries and sequenced. One of these mutants proved to be a 20 base-pair deletion formed between two short (3 base-pair) direct repeat sequences, while the second was the result of a 58 base-pair insertion accompanied by a 13 base-pair deletion.

Characterization of mutations induced by 2-(N-acetoxy-N-acetyl)aminofluorene in the dihydrofolate reductase gene of cultured hamster cells.

To determine the types of alterations in gene structure that are induced by the carcinogen 2-(N-acetoxy-N-acetyl)aminofluorene, we used this compound to generate mutations at the dihydrofolate reductase (DHFR) locus (DHFR) in Chinese hamster ovary cells. Twenty-nine independent enzyme-deficient mutants were isolated. A profile of the 26-kilobase (kb)-long gene was obtained by Southern blot analysis of the mutant and parental DNAs digested with BstEII/Kpn I. Hybridization to a mixed probe of 10 DHFR genomic and cDNA fragments revealed 12 bands that scan 34 kb. Twenty-one DHFR- clones (72%) contained small mutations (changes less than 100 base pairs in size). Large or small deletions involving various parts of the gene occurred in eight of the mutants (28%). A large deletion (greater than 35 kb) with 5' and 3' breakpoints mapping to approximately the same location was noted in four mutants. One mutant has undergone a deletion of 550-900 bp that eliminated the first coding exon. Concomitantly, a chromosomal event (either translocation, insertion, or inversion) has separated the 5' flank from the body of the gene. In another mutant, four deletions have occurred at the DHFR 5' end and internally. Restriction fragment length polymorphism analysis of the mutant DNAs with exon-specific probes localized three mutations. One mutant has lost a Taq I (TCGA) site, and another has lost a Sac I (GAGCTC) site. In a third, a GC----TA transversion has created a BstEII (GGTNACC) site. Finally, we used HPLC to determine the ratio of acetylated (12%) to deacetylated (88%) 2-aminofluorene adducts formed in the parental cells. A correlation between the mutational specificities and the conformational changes induced by the two types of DNA adducts is discussed.

Spontaneous deletion formation at the aprt locus of hamster cells: the presence of short sequence homologies and dyad symmetries at deletion termini.

To examine the factors governing the generation of DNA sequence rearrangements in mammalian somatic cells, we have cloned and sequenced novel junctions produced by six spontaneous deletion mutations at the aprt locus of Chinese hamster ovary cells. Our analyses indicate that these rearrangements were produced by non-homologous recombinational events occurring between short (2-7 bp) sequence repeats at the two termini of the deletion which leave one copy of the repeat in the mutant gene. Certain tri- and tetranucleotides recur at the deletion termini, suggesting that these may possibly be a recognition sequence for an enzyme involved in the event. No other gene structural alterations were found at the novel junctions or in neighbouring sequences. The deletions are not randomly distributed over the aprt gene; four termini clustered in a 40-bp sequence. This region of aprt is unusual as it contains both significant stretches of dyad symmetry which could potentially form stable DNA secondary structures and short direct repeats. Regions of dyad symmetry were also found at at least one terminus of all the deletions. In view of the similar properties of this set of deletions, possible mechanisms for the formation of this type of gene rearrangement are considered.

Use of T-cell receptor gene probes to quantify the in vivo hprt mutations in human T-lymphocytes

T-cell receptor (Ti) gene restriction fragment patterns (RFPs) were determined by Southern blots of genomic DNA obtained from T-lymphocyte colonies isolated from a single normal individual. 4 wild-type colonies and 11 in vivo derived 6-thioguanine-resistant mutant colonies with previously characterized hprt gene structural alterations were studied. Among the hprt mutants, 10 of the 11 showed unique Ti RFPs indicating their origins in different in vivo progenitors. Unique Ti RFPs were also seen among the wild-type T-cell colonies. One, however, shared its Ti RFP with a mutant. These results suggest that mutation in vivo of the hprt gene in human T-lymphocytes occurred after thymic maturation and that the 11 recovered hprt mutants probably resulted from 11 independent mutational events.

Transformation of NIH 3T3 cells by microinjection of Ha-ras p21 protein.

Alteration in gene structure has been shown to occur in some human tumours. These altered genes, termed oncogenes, were originally identified by their ability to induce foci of transformed cells on transfected mouse 3T3 cultures. The oncogene identified in the EJ/T24 human bladder carcinoma is similar to the transforming gene of BALB and Harvey murine sarcoma virus (MSV) and differs from its counterpart in normal cells by a single amino acid. All three of these Ha-ras genes direct the production of similar proteins (p21). While the ras gene appears to be involved in tumour formation in some situations, its role is unclear. The ras protein product (p21) binds guanine nucleotides and has a unique autophosphorylating activity, but no other enzymatic activity has been found. We report here the injection of purified Ha-ras p21 protein, made in Escherichia coli from the gene of BALB-MSV, into NIH 3T3 cells and show that the purified protein itself is sufficient to induce a transformed morphology. In addition, the injected protein stimulates quiescent cells to enter the S-phase of the cell cycle. This result clearly demonstrates that the ras gene functions directly through the protein product. It also establishes an assay for the protein which depends on its activity within a living cell. The transforming activity of a p21 ras protein equivalent to the product of the normal cellular ras gene, is also demonstrated.

Structure of mutant alleles at the aprt locus of Chinese hamster ovary cells

To determine the types of gene structural alterations causing deficiency of adenine phosphoribosyl transferase (aprt) activity in spontaneous and chemically induced mutations of cultured somatic cells, we analyzed the restriction enzyme cleavage patterns of aprt gene sequences in mutant strains selected from Chinese hamster ovary cells. Patterns of aprt-containing fragments in Southern blots were mostly unchanged in our collection of 280 ethyl methane sulfonate-induced and spontaneous aprt- mutants, suggesting that base-pair changes or other alterations below our limit of resolution on agarose gels (approximately 50 base-pairs) are responsible for the great majority of mutations at the aprt locus. Occasionally, these mutations could be localized when they resulted in the loss or gain of a restriction enzyme site and the generation of new fragments of predictable size. Deletions of aprt-containing sequences were detected in only eight of 119 spontaneous mutants and in only one ethyl methane sulfonate-induced mutant. An insertion of 300 base-pairs near the 5' end of the aprt structural gene was found in one spontaneous aprt- strain. This insertion mutant was stable with a reversion frequency of less than 2 X 10(-7). Several unstable aprt- mutants were detected in our collection, but these had no observable alterations of aprt coding or flanking sequences.

Molecular analysis of argininosuccinate synthetase deficiency in human fibroblasts.

We have analyzed cultured skin fibroblasts derived from patients with argininosuccinate synthetase deficiency for alterations in gene structure, mRNA content, and protein structure. Genomic DNA was digested with the endonucleases EcoRI or HindIII, and the fragments were analyzed by Southern blotting and hybridization with a cDNA probe for argininosuccinate synthetase. The blot pattern is complex because there are at least 10 copies of argininosuccinate synthetase-like genes scattered over multiple human chromosomes. All nine patients studied showed patterns of DNA fragments that were indistinguishable from the normal control cell lines, and despite the possibility that the complexity could mask some changes, major deletions of the active gene(s) were not present. Blot hybridization of RNA indicated the presence of hybridizable mRNA of approximately normal size in seven of seven individuals examined with a suggestion of some heterogeneity. Analysis of enzyme antigen by protein transfer from NaDodSO4 containing polyacrylamide gels revealed considerable heterogeneity. This analysis revealed no cross-reacting material (CRM) in nine cell lines, CRM of normal molecular weight in one cell line, and CRM of reduced molecular weight in one cell line. These findings suggest that the genes for argininosuccinate synthetase in most citrullinemia patients are transcribed and produce stable mRNA. These mRNA either are not translated, or the translation product (enzyme) is rapidly degraded or is immunologically nonreactive. Defective gene expression in this disorder appears to involve abnormal mRNA, which may be altered by point mutations, frame shift mutations, deletions, insertions or particularly by abnormal RNA processing.

Alterations of Gene Structure in Ethyl Methane Sulfonate-Induced Mutants of Mammalian Cells

To determine the types of alterations in gene structure induced by DNA-alkylating agents, we analyzed the restriction enzyme cleavage patterns of adenine phosphoribosyltransferase gene sequences in mutant strains of Chinese hamster ovary cells deficient in this enzyme. Base pair changes as detected by loss of restriction enzyme sites were found, but no major internal gene rearrangements could be detected.

Alterations of gene structure in ethyl methane sulfonate-induced mutants of mammalian cells.

To determine the types of alterations in gene structure induced by DNA-alkylating agents, we analyzed the restriction enzyme cleavage patterns of adenine phosphoribosyltransferase gene sequences in mutant strains of Chinese hamster ovary cells deficient in this enzyme. Base pair changes as detected by loss of restriction enzyme sites were found, but no major internal gene rearrangements could be detected.

Bone morphodifferentiation and tumorigenesis.

BONE MORPHODIFFERENTIATION AND TUMORIGENESIS* MARSHALL R. URISTf By means of correlated observations on the roentgenographic and histological structure, in an article written over 50 yr ago, Phemister [1] demonstrated diat osteosarcomas contain tumorous and normal nontumorous bone. Normal growing bone has a trabecular pattern that is specific for each bone and part of a bone as well as a characteristic microscopic palisade of cuboidal basiphilic cells named osteoblasts. Tumorous bone has a disoriented structure widi trabeculae enveloped in a random arrangement of bizarrely shaped anaplastic cells. Tumorous bone grows by proliferation of transformed osteoblasts. The alterations in gene structure and function in the transformed state are obscure, but the involvement is comparable to a mutation. The criteria for cell transformation generally include: loss of cell contact inhibition; altered morphology ; increased growdi rate; increased capacity to persist in serial subcultures; chromosomal abnormalities; increased resistance to reinfection by die transforming virus; emergence of new antigens; capacity to form neoplasms [2]. Viral-induced malignant transformation constitutes a heritable change and is accompanied by the loss of regulatory controls of cell growdi. Present knowledge ofnonmalignant morphodifferentiation, as well as malignant transformation, is chiefly descriptive. Data on the biochemical controls of morphodifferentiation are lacking. The process beginning widi the fertilized ovum and ending widi the mechanics of unfolding of die structural characteristics, form, and size of functional tissues defies present understanding. The problem is the prerogative of students of dieoretical, as distinguished from experimental, biology. ?The 27th Dallas B. Phemister Lecture, November 15, 1977. tDirector, UCLA Bone Research Laboratory, 1000 Veteran Avenue, Los Angeles, California 90024. 1 am deeply indebted to my collaborators, D. H. Hanamura, N. Nakata, J. Felser, G. Styfestad, M. Miki, J. Mirra, K. Tibone, and G.A.M. Finerman and to S. Kirkpatrick, S. Uyeno, and E. King for technical assistance in securing die information presented in this paper. The research was supported by a grant-in-aid from USPHS, National Institutes of Health, NIDR, DE-02103-14.© 1979 by The University of Chicago. 0031-5982/79/2222-0008$01.00 Perspectives in Biology andMedicine ¦ Winter 1979 ¦ Part 2 | S89 Bone morphodifferentiation can be consistently initiated in postfetal life in muscle, a tissue that in die lifetime of the animal does not spontaneously differentiate into any odier organ structure [3]. As in development in embryonic life, the process can be arbitrarily divided into morphogenetic and cytodifferentiation phases [4]. The morphogenetic phase begins by a chain of reactions of proliferating cell populations to chemical agents collectively termed morphogens [5-8]. Every new generation of cells synthesizes and transfers morphogen in minute quantities to a succeeding generation ofresponding tissue cells. Theoretically, morphogens have as low molecular mass as hydrophobic peptides or polypeptides [7], which are chiefly bound to proteins and only partly free or diffusible. The concentration of free morphogen in developing tissues may be as low as ??-10 M [9]. The morphogen concept is a modification of die Spemann theory of embryonic inducer substances and die Waddington tiieory of embryonic evocators [5]. Cells exposed to the critical concentration of free morphogen in the extracellular fluid differentiate or adopt a new padiway of development of a specialized form [7]. Covert stages of differentiation of skeletal tissues are observed by accumulation of biosyndietic products such as hyaluronate, collagen, and enzymes (e.g., hyaluronidase, alkaline phosphatase ). Overt stages are recognized by deposition of extracellular hyaline matrix or calcifiable bone collagen matrix. Nodiing is known about die biochemical reactions leading to the development of a concentration gradient between die source of die morphogen and its disposition in a population sink of competent responding cells. For a true comprehension of the mode of action of morphogens, present concepts of the action of hormones on specialized target cells are inadequate. Morphogens are much lower in concentration in interstitial fluids than hormones. Morphogens are surface oriented in highly compartmentalized intra- and extracellular fluids and released from protein-protein complexes. Transfer of morphogen from a transport system to a plasma-membrane morphogen receptor molecule would release a gene regulator molecule which in turn could de-repress a specific form of gene expression. An effective concentration ofmorphogen develops when a population of proliferating competent cells reaches a critical mass. The population...