Malignancy and transformation: expression in somatic cell hybrids and variants.
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Animals↗
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The relative levels of different types of beta-mRNA and beta-globin in BFU-E derived colonies from patients with beta chain variants; further evidence for somatic mosaicism in the Hb Costa Rica carrier [beta 77(EF1)His-->Arg].
We have identified and quantitated the different types of mRNA in single BFU-E derived colonies from Hb S and Hb Atlanta [beta 75 (E19)Leu-->Pro] heterozygotes and observed that the normal and mutated mRNAs were present in equal quantities. Similar studies for the different protein products gave less accurate data because high performance liquid chromatography methods were not sensitive enough for the analysis of a single colony, and as many as five colonies needed to be combined. The level of Hb S (approximately 40%) was the same as in red cell lysates of the Hb S heterozygote, while that of the unstable beta-Atlanta chain was lower than expected from the values observed in red cells. Similar studies for a carrier of the stable Hb Costa Rica [beta 77(EF1) His-->Arg] which was reported to be the result of a somatic cell mutation (1) gave quite different data. Dot-blot analysis with 32P-labeled probes and allele specific amplification methodology identified numerous colonies with beta A-mRNA only, while 12-15% of the colonies contained both beta A- and beta-Costa Rica-mRNA. This limited distribution of the beta-Costa Rica-mRNA was confirmed by hemoglobin analysis with anion exchange high performance liquid chromatography. These results are considered to provide additional and convincing evidence for a somatic mosaicism for the CAC-->CGC mutation at codon 77 of the beta gene which occurred during the development of the embryo and results in the presence of only some 6-8% of the abnormal Hb Costa Rica in the circulating red cells.
Erythroid Precursor Cells↗
[Adolescent somatic development and its variants].
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Adolescent↗
Linker histones play a role in male meiosis and the development of pollen grains in tobacco.
To examine the function of linker histone variants, we produced transgenic tobacco plants in which major somatic histone variants H1A and H1B were present at approximately 25% of their usual amounts in tobacco chromatin. The decrease in these major variants was accompanied by a compensatory increase in the four minor variants, namely, H1C to H1F. These minor variants are smaller and less highly charged than the major variants. This change offered a unique opportunity to examine the consequences to a plant of major remodeling of its chromatin set of linker histones. Plants with markedly altered proportions of H1 variants retained normal nucleosome spacing, but their chromosomes were less tightly packed than those of control plants. The transgenic plants grew normally but showed characteristic aberrations in flower development and were almost completely male sterile. These features correlated with changes in the temporal but not the spatial pattern of expression of developmental genes that could be linked to the abnormal flower phenotypes. Preceding these changes in flower morphology were strong aberrations in male gametogenesis. The earliest symptoms may have resulted from disturbances in correct pairing or segregation of homologous chromosomes during meiosis. No aberrations were observed during mitosis. We conclude that in plants, the physiological stoichiometry and distribution of linker histone variants are crucial for directing male meiosis and the subsequent development of functional pollen grains.
Chromatin↗
Genetic analysis of a transcriptional activation pathway by using hepatoma cell variants.
A hierarchy of liver-enriched transcription factors plays an important role in activating expression of many hepatic genes. In particular, hepatocyte nuclear factor 4 (HNF-4) is a major activator of the gene encoding HNF-1, and HNF-1 itself activates expression of more than 20 liver genes. To dissect this activation pathway genetically, we prepared somatic cell variants that were deficient in expression of the liver-specific alpha 1-antitrypsin (alpha 1AT) gene, which requires both HNF-1 and HNF-4 for high-level gene activity. This was accomplished in two steps. First, hepatoma transfectants that stably expressed two selectable markers under alpha 1AT promoter control were prepared; second, variant sublines that could no longer express either transgene were isolated by direct selection. In this report, we demonstrate that the variants contain defects in the HNF-4/HNF-1 activation pathway. These defects functioned in trans, as expression of many liver genes was affected, but the variant phenotypes were recessive to wild type in somatic cell hybrids. Three different variant classes could be discriminated by their phenotypic responses to ectopic expression of either HNF-4 or HNF-1. Two variant clones appeared specifically deficient in HNF-4 expression, as transfection with an HNF-4 expression cassette fully restored their hepatic phenotypes. Another line activated HNF-1 in response to forced HNF-4 expression, but activation of downstream genes failed to occur. One clone was unresponsive to either HNF-1 or HNF-4. Using the variants, we demonstrate further that the chromosomal genes encoding alpha 1AT, aldolase B, and alpha-fibrinogen display strict requirements for HNF-1 activation in vivo, while other liver genes were unaffected by the presence or absence of HNF-1 or HNF-4. We also provide evidence for the existence of an autoregulatory loop in which HNF-1 regulates its own expression through activation of HNF-4.
Animals↗
[Nature of the phenotypic variability of somatic cells in culture: unstable phenotypic changes].
It was stated elsewhere ( Glebov , Abramyan , 1983) that the appearance of a number of phenotypic variants detected in somatic cell populations with high frequency should be provided by genetical unstable alterations. The properties of somatic cell variants that reproduce unstably a changable phenotype in the course of cell generations are analysed. These variants: (1) appear accidentally and independently on selectivity agent; (2) as a rule, the frequency of the variant arising does not increase under the action of mutagens; (3) the phenotypic reversion of unstable variants is a stochastic process; (4) such variants are characterized by intraclonal heterogeneity and by the segregation of stable alternative variants. The number of properties of phenotypically unstable variants isolated by one-step selection is similar to those for somatic cell variants isolated in the course of multistep selection. The latter are characterized by phenotypic reversion too. The appearance of unstable phenotypic variants is concluded to be associated with the genetical unstable alterations. It is argued that at least part of above alterations should be induced by the insertion of mobile genetic elements. The features of karyotypical variation in somatic cell population allow to conclude that the karyotype of cultured somatic cells is a genetically unstable attribute. The features mentioned above are: a high frequency of karyotypical alterations which is inherited by the cells with difference in the frequency of arising of karyotypical alteratons . The unstability of karyotype is restricted to the genetic unstability that is seen from non-random karyotypic variation, and interclonal difference in the chromosome stability. The site-specificity of karyotype alterations that proceed with high frequency allow to put forward a hypothesis that the process of mobile genetic element transposition is induced on the early stages of the history of constant cultured cell lines.
Animals↗
Isolation of myeloma variants with predefined variant surface immunoglobulin by cell sorting.
We describe a procedure for the isolation of somatic cell variants in which gene products are expressed on the cell surface that are not expressed in the wild type. Cloned cells of the myeloma line MPC 11, which expresses an IgG2b protein, were incubated with an antiserum specific for IgGI and IgG2a. Cells reacting with this antiserum were stained with a fluorescent anti-antiserum and enriched in three cycles of sorting in the fluorescence-activated cell sorter and subsequent growth in vitro. From the enriched population two variants were isolated by cloning in soft agar. One of them expressed a variant immunoglobulin that types serologically as an IgG2a but whose variable portion was idiotypically related to that of the MPC 11 wild-type protein.
B-Lymphocytes↗
Differential Expression of Histone H3 Gene Variants during Cell Cycle and Somatic Embryogenesis in Alfalfa.
Northern analysis has revealed substantial differences in mRNA accumulation of the two histone H3 gene variants represented by pH3c-1 and pH3c-11 cDNA clones. Both in partially synchronized cell suspension cultures and in protoplast-derived cells from alfalfa, Medicago varia, the maximal level of the histone H3-1 gene transcript coincided with the peak in [(3)H]thymidine incorporation. Histone H3-11 mRNA was detectable in cells throughout the period of the cell cycle studied. Various stress factors such as medium replacement, enzyme digestion of the cell wall, osmotic shock, and auxin treatment considerably increased the level of the histone H3-11 transcript. In alfalfa (Medicago sativa), the presence of H3-11 mRNA in unorganized tissues of microcallus suspension and in somatic embryos induced by auxin treatment supports the idea that this H3 variant exists in a continously active state of transcription. During embryo development, the early globular stage embryos showed increased accumulation of histone H3-11 mRNA in comparison with the later stages. The highest level of the histone H3-1 transcript was detectable 1 day after treatment of callus tissues with 2,4-dichlorophenoxyacetic acid. Somatic embryos contained appreciable levels of histone H3-1 transcripts at all stages of somatic embryo development. These observations suggest that the histone H3-1 gene belòngs to the class of replication-dependent histone genes. The histone H3-11 gene showed characteristics of a constitutively expressed replacement-type histone gene, with a specific characteristic that external factors can influence the level of gene transcription.
Journal Article↗
Colicine K. V. The somatic antigen of a non-colicinogenic variant of E. coli K235.
The somatic antigen of the non-colicinogenic bacillus E. coli K235 L(-)OC(-) has been isolated, and its chemical and serological properties have been compared with those of colicine K. The antigen of the non-colicinogenic bacillus has a protein content significantly lower than that of the C(+) antigen, a difference which might be related to the antibacterial activity of the latter. The lipocarbohydrate components of the two antigens are chemically very similar; both contain the same proportions of galactose, glucose, heptose, rhamnose, glucosamine, and mannosamine. When tested by agar diffusion, the two antigens are indistinguishable, as are their lipocarbohydrate components. Our studies indicate that the bactericidal activity of colicine K does not reside in its lipocarbohydrate but in its protein component.
Anti-Bacterial Agents↗
A Comprehensive Bioinformatics Approach to Analysis of Variants: Variant Calling, Annotation, and Prioritization.
Next-Generation Sequencing (NGS), also known as high-throughput sequencing technologies, has enabled rapid and efficient sequencing of large amounts of DNA and RNA. These technologies have revolutionized the field of genomics, transcriptomics, and proteomics and have been widely used in cancer research, leading to advances in clinical diagnosis and treatment. Improvements in the NGS technologies enabled millions of fragments to be sequenced simultaneously in a time- and cost-effective manner and resulted in large amount of genomic data which require efficient analysis methods. Analysis of the genomic data requires both efficient computer resources and bioinformatics approaches. This chapter details a comprehensive computational approach and analysis steps for genomic data analysis.
Computational Biology↗
Isolation of a cloned cell line expressing variant H-2Kk using fluorescence-activated cell sorting.
We describe here a method for the isolation of somatic cell variants that express a structurally altered gene product on their cell surface. The method makes it possible to select positively for cells of the variant phenotype which is defined by loss of a given antigenic determinant. As an example, we present the isolation of a cloned cell line expressing a structurally altered H-2Kk molecule, designated H-2KkS1. Cells of this phenotype were found to be present in the wild-type population at a frequency of 10(-5)-10(-6) and were isolated in three rounds of selection in the fluorescence-activated cell sorter (FACS) and subsequent growth in vitro.
Animals↗
Isolation of four core histones from human sperm chromatin representing a minor subset of somatic histones.
Using high performance liquid chromatography we have successfully purified four core histones from mature human sperm chromatin. The H2A variants present in sperm (H2A.X and limited H2A.Z) have been shown previously to be minor variants in somatic chromatin. The histones are highly modified as evidenced by extensive acetylation and an as yet uncharacterized multicharge modification of H2B. Based on our data, we conclude that histone proteins are a minor component of each mature spermatozoa. Given the unique nature of the histone variants present in sperm, we propose that this chromatin component has a specific function and may possibly facilitate the programming of genes which will be active in early development.
Amino Acids↗
Visceral pain.
Visceral pain is the most common form of pain produced by disease and one of the most frequent reasons why patients seek medical attention. Yet much of what we know about the mechanisms of pain derives from experimental studies of somatic not visceral nociception. The conventional view is that visceral pain is simply a variant of somatic pain, a view based on the belief that a single neurological mechanism is responsible for all pain. However, the more we learn about the mechanisms of somatic and visceral pain, the more we realise that although these two processes have much in common, they also have important differences. Although visceral pain is an important part of the normal sensory repertoire of all human beings and a prominent symptom of many clinical conditions, not much clinical research has been done in this field and there are few clinical scientists with expertise in the management of visceral pain. Instead, visceral pain is usually treated by a range of specialists who take quite different approaches to the management of this type of pain. Thus, the management of visceral pain is frequently unsatisfactory. In this review, we consider visceral pain as a separate form of pain and examine its distinct sensory properties from a clinical perspective. We describe recent research findings that may change the way we think about visceral pain and, more importantly, may help develop new procedures for its management.
Humans↗
Somatic mitochondrial DNA (mtDNA) mutations in papillary thyroid carcinomas and differential mtDNA sequence variants in cases with thyroid tumours.
Somatic mutations in mtDNA have recently been identified in colorectal tumours. Studies of oncocytic tumours have led to hypotheses which propose that defects in oxidative phosphorylation may result in a compensatory increase in mitochondrial replication and/or gene expression. Mutational analysis of mtDNA in thyroid neoplasia, which is characterised by increased numbers of mitochondria and is also one of the most common sites of oncocytic tumours. has been limited to date. Using the recently developed technique of two-dimensional gene scanning, we have successfully examined 21 cases of thyroid tumours, six cases of non-neoplastic thyroid pathology, 30 population controls, nine foetal thyroid tissues and nine foetal tissues of non-thyroid origin, either kidney or liver. We have identified three different somatic mutations (23%) in papillary thyroid carcinomas. In addition, we have found significant differential distributions of mtDNA sequence variants between thyroid carcinomas and controls. Interestingly, these variants appear to be more frequent in the genes which encode complex I of the mitochondrial electron transport chain compared to normal population controls. These findings suggest first, that somatic mtDNA mutations may be involved in thyroid tumorigenesis and second, that the accumulation of certain non-somatic variants may be related to tumour progression in the thyroid.
Adenoma↗
Somatic mutations of WNT/wingless signaling pathway components in primitive neuroectodermal tumors.
Primitive neuroectodermal tumors (PNETs) represent the most frequent malignant brain tumors in childhood. The majority of these neoplasms occur in the cerebellum and are classified as medulloblastomas (MB). Most PNETs develop sporadically; however, their incidence is highly elevated in patients carrying germline APC gene mutations. The APC gene encodes a central component of the WNT/wingless developmental signaling pathway. It regulates the levels of cytoplasmic beta-catenin protein that plays a central role in neural development and cell proliferation. We analyzed 87 sporadic PNETs and 10 PNET cell lines for mutations of the APC gene and beta-catenin (CTNNB1) gene using single strand conformational polymorphism (SSCP) and sequencing analysis. We examined the mutation cluster region of APC (codons 1255--1641) for germline variants and somatic mutations. The medulloblastoma cell line MHH-MED-2 carried a Glu1317Gln missense germline variant and a sporadic MB sample showed a somatic Pro1319Leu substitution. Mutational analysis of exon 3 of CTNNB1 uncovered 4 PNETs (4.8%) with somatic missense mutations. These mutations caused amino acid substitutions in 3 of 80 medulloblastomas (Ser33Phe, Ser33Cys and Ser37Cys) and 1 of 4 supratentorial PNETs (Gly34Val). All mutations affected GSK-3 beta phosphorylation sites of the degradation targeting box of beta-catenin and resulted in nuclear beta-catenin protein accumulation. Deletions of CTNNB1 were not detected by PCR amplification with primers spanning exons 1--5. Our data indicate that inappropriate activation of the WNT/wingless signaling pathway by mutations of its components may contribute to the pathogenesis of a subset of PNETs.
Adenomatous Polyposis Coli Protein↗
Mitotic recombination produces the majority of recessive fibroblast variants in heterozygous mice.
Mice heterozygous at Aprt (adenine phosphoribosyltransferase) were used as a model to study in vivo loss of heterozygosity (LOH) in normal fibroblasts. Somatic cell variants that exhibited functional loss of the wild-type Aprt in vivo were recovered as APRT-deficient cell colonies after culturing in selection medium containing 2, 6-diaminopurine (DAP), an adenine analog that is toxic only to cells with APRT enzyme activity. DAP-resistant (DAP(r)) fibroblast variants were recovered at a median frequency of 12 x 10(-5) from individual ears from progeny of crosses between mouse strains 129/Sv and C3H/HeJ. The frequency of DAP(r) variants varied greatly among individual ears, suggesting that they preexisted in vivo and arose at various times during development. Polymorphic molecular markers and a cytological marker on the centromere of chromosome 8 made it possible to discriminate between each of six possible mechanistic pathways of LOH. The majority (about 80%) of the DAP(r) variants were a consequence of mitotic recombination. The prevalence of mitotic recombination in regions proximal to Aprt did not correlate with meiotic map distances. In particular, there was a higher than expected frequency of crossovers within the interval 59 cM to 67 cM. The high spontaneous frequency of Aprt LOH, mediated primarily by mitotic recombination, is fully consistent with our previous results with human peripheral T cells from individuals known to be heterozygous at APRT. Thus, this Aprt heterozygote mouse is a valid model for studying somatic mutagenesis and mitotic recombination in vivo.
Adenine Phosphoribosyltransferase↗
Inherited variants of MYH associated with somatic G:C-->T:A mutations in colorectal tumors.
Inherited defects of base excision repair have not been associated with any human genetic disorder, although mutations of the genes mutM and mutY, which function in Escherichia coli base excision repair, lead to increased transversions of G:C to T:A. We have studied family N, which is affected with multiple colorectal adenomas and carcinoma but lacks an inherited mutation of the adenomatous polyposis coli gene (APC) that is associated with familial adenomatous polyposis. Here we show that 11 tumors from 3 affected siblings contain 18 somatic inactivating mutations of APC and that 15 of these mutations are G:C-->A transversions--a significantly greater proportion than is found in sporadic tumors or in tumors associated with familial adenomatous polyposis. Analysis of the human homolog of mutY, MYH, showed that the siblings were compound heterozygotes for the nonconservative missense variants Tyr165Cys and Gly382Asp. These mutations affect residues that are conserved in mutY of E. coli (Tyr82 and Gly253). Tyrosine 82 is located in the pseudo-helix-hairpin-helix (HhH) motif and is predicted to function in mismatch specificity. Assays of adenine glycosylase activity of the Tyr82Cys and Gly253Asp mutant proteins with 8-oxoG:A and G:A substrates show that their activity is reduced significantly. Our findings link the inherited variants in MYH to the pattern of somatic APC mutation in family N and implicate defective base excision repair in predisposition to tumors in humans.
Amino Acid Sequence↗
Molecular loss variants of the murine major histocompatibility complex: nonexpression of H-2K antigens associated with marked reduction in H-2K mRNA as determined by oligonucleotide hybridization analysis.
Somatic cell variants of the murine major histocompatibility complex were isolated to study the molecular features required for H-2K gene expression. In vitro selection was performed on a heterozygous [H-2b (Kb,Db) X H-2d (Kd, Dd, Ld)] pre-B lymphoblastoid cell line (R8) for variants that had lost membrane expression of the H-2Kb gene product. Analysis of a number of independently isolated variant cell lines by cytofluorometry with monoclonal antibodies to the Kb, Kd, Db, and Ld antigens revealed a variety of H-2 phenotypes. Variants were classified as either molecular loss for those that had lost K antigen expression only or as haplotype loss for those that no longer expressed the entire H-2b haplotype (i.e., negative for Kb and Db). DNA hybridization analysis with a K gene-specific oligonucleotide indicated that the Kb gene was present in all of the molecular loss variants, suggesting that gene deletion was not responsible for the loss of Kb antigen expression. In contrast, the Kb gene was not detected in haplotype loss variants. For analyzing the mutants at the RNA level, hybridization with H-2-specific synthetic oligonucleotides provided a definitive procedure to identify specific class I gene transcripts in the H-2 heterozygous cell lines. Such analyses were performed on the molecular loss variants and revealed that in a subset of variants (R8.2, R8.96, R8.116, R8.178) there was an absence of Kb mRNA. Kd mRNA was identified for all but one (R8.2) of the Kb mRNA-deficient cell lines, a finding consistent with the serotype assigned to each. Transcription of the linked Db gene was normal. These data demonstrate that a specific alteration for the K gene in several independently selected cell lines gave rise to the altered H-2 phenotype. Such variants offer the potential to analyze the properties of the mammalian genome responsible for controlling expression of individual members of the major histocompatibility complex multigene family.
Animals↗