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PubMed2001

Drosophila male meiosis as a model system for the study of cytokinesis in animal cells.

Drosophila male meiosis offers unique opportunities for mutational dissection of cytokinesis. This system allows easy and unambiguos identification of mutants defective in cytokinesis through the examination of spermatid morphology. Moreover, cytokinesis defects and protein immunostaining can be analyzed with exquisite cytological resolution because of the large size of meiotic spindles. In the past few years several mutations have been isolated that disrupt meiotic cytokinesis in Drosophila males. These mutations specify genes required for the assembly, proper constriction or disassembly of the contractile ring. Molecular characterization of these genes has identified essential components of the cytokinetic machinery, highlighting the role of the central spindle during cytokinesis. This structure appears to be both necessary and sufficient for signaling cytokinesis. In addition, many data indicate that the central spindle microtubules cooperatively interact with elements of the actomyosin contractile ring, so that impairment of either of these structures prevents the formation of the other.

Animals↗
PubMed2005

Reliable low-density DNA array based on allele-specific probes for detection of 118 mutations causing familial hypercholesterolemia.

BACKGROUND: Patients with familial hypercholesterolemia (FH) have a high risk of premature cardiovascular disease (PCVD). Mutations in the LDL receptor (LDLR) gene and the R3500Q mutation in the apolipoprotein B (APOB) gene are known to cause FH, but lack of high-throughput methods makes routine genetic diagnosis difficult. The objective of this work was to develop a DNA array for large-scale identification of mutant LDLR alleles. METHODS: We developed a low-density oligonucleotide microarray to identify 118 DNA sequence variations (117 for the LDLR gene and 1 for the APOB gene). We verified specificity and sensitivity by analyzing 1180 previously sequenced DNA samples, and conducted a blind study screening 407 Spanish patients with a clinical diagnosis of FH. RESULTS: The DNA array confirmed the previous genotyping results in almost all cases. In the blind study, the microarray detected at least 1 mutation in 51% of the patients for whom clinical diagnosis was classified as certain according to Dutch FH-MEDPED criteria; it also identified mutations in 37% of those with a diagnosis of probable/possible FH, thus giving a definite diagnosis. Patients harboring null mutations had shorter PCVD-free survival times and higher relative risk of PCVD than patients with a missense mutation. CONCLUSIONS: The proposed DNA array allows large-scale population screening and provides molecular information regarding mutation type and its correlation with clinical severity of FH, which can be used to develop therapeutic strategies.

Alleles↗
PubMed1999

Multiple endocrine neoplasia type 1.

Combined clinical and laboratory investigations of multiple endocrine neoplasia type 1 (MEN1) have resulted in an increased understanding of this disorder which may be inherited as an autosomal dominant condition. Defining the features of each disease manifestation in MEN1 has improved patient management and treatment, and has also facilitated a screening protocol to be instituted. The application of the techniques of molecular biology has enabled the identification of the gene causing MEN1 and the detection of mutations in patients. The function of the protein encoded by the MEN1 gene has been shown to be in the regulation of JunD-mediated transcription but much still remains to be elucidated. However, these recent advances provide for the identification of mutant MEN1 gene carriers who are at a high risk of developing this disorder and thus require regular and biochemical screening to detect the development of endocrine tumours.

Genes, Tumor Suppressor↗
PubMed2003

Dictyostelium discoideum transformation by oscillating electric field electroporation.

Dictyostelium discoideum has been used as a genetically tractable model organism to study many biological phenomena. High-efficiency transformation is a prerequisite for successful genetic screens such as mutant complementation, identification of suppressor genes, or insertional mutagenesis. Although exponential decay electroporation is the standard transformation technique for D. discoideum, its efficiency is relatively low and its reproducibility is weak. Here we optimized the oscillating electroporation technique for D. discoideum transformation and compared it to the exponential decay electroporation. A 20-fold increase in the efficiency was resproducibly achieved. This alternative electroporation technique should facilitate future genetic approaches in D. discoideum.

Adaptation, Physiological↗
PubMed2000

Multiple endocrine neoplasia type 1: from bedside to benchside.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the combined occurrence of parathyroid, pancreatic endocrine, and anterior pituitary tumors. MEN1 has two characteristics; a hormone excess and a sometimes lethal outcome due to malignant tumors. The recent identification of the MEN1 gene has opened the door to a much deeper understanding of this syndrome. Germline MEN1 mutations have been identified in most MEN1 families. They were not found, however, in families with familial pituitary tumors. Thus, studies with the MEN1 gene helped to establish that mutation of some other gene(s) is likely causative of the MEN1 phenocopy. These recent advances provide for the identification of mutant MEN1 gene carriers who are at a high risk of developing MEN1. The protein encoded by the MEN1 gene has been shown to function in the regulation of JunD-activated transcription but much still remains to be elucidated.

Genes, Tumor Suppressor↗
PubMed2000

[Multiple endocrine neoplasia: a clinical model for applying molecular genetic techniques].

Multiple endocrine neoplasias (MEN) are syndromes inherited as autosomal dominant. The application of the techniques of molecular biology has made possible the identification of the genes causing MEN 1 and 2. The gene responsible for MEN 1 belongs to the family of tumor suppressor genes and encodes for a protein named MENIN whose function remains to be elucidated. The identification of mutant MEN 1 gene carriers who are at risk of developing this syndrome requires frequent biochemical screening for the development of endocrine tumors. MEN 2 is a consequence of mutations in the Ret proto-oncogene (c-Ret). This gene encodes for a tyrosine kinase receptor thought to play a role in the development of neural crest-derived tissue. Members of kindred with either MEN 2A or MEN 2B should be screened by direct DNA testing early in life for mutations in c-Ret. Those with the mutation should be advised to have thyroidectomy at five years of age in children with MEN 2A and earlier in children with MEN 2B. Some cases of sporadic MTC are actually MEN 2A or Familial MTC after c-Ret testing is done, therefore routine application of this test is recommended in all cases of apparent sporadic MTC.

Adult↗
PubMed2001

McCune-Albright syndrome: radiological and MR findings.

McCune-Albright syndrome (MAS) is a non-inherited disorder due to the GNAS1 gene mutation. The syndrome is characterized with the triad of polyostotic fibrous dysplasia, pigmented skin lesions, endocrinopathy, and precocious puberty. We report the case of a 14-year-old boy, presenting with sclerotic type of polyostotic fibrous dysplasia. Radiological methods including plain X-ray film, MR and whole body bone scintigraphy suggested the diagnosis of MAS. MRI provided more directly perceived images and it was more sensitive in demonstrating the lesion: its shape, contents, especially the size of the affected region. Histopathological study and the identification of mutant gene finally confirmed the diagnostic result.

Adolescent↗
PubMed2006

Physiological and biochemical characteristics and molecular-biological identification of a gigantic tobacco mutant.

A new gigantic late-flowering tobacco mutant was isolated in tobacco field in Xunyang county, Shaanxi province, in 2001. It was rescued through tissue culture and a large amount of regenerated plantlets were obtained. The results of the comparison between the regenerated plants from this mutant and the wild type plant (K346) were as follows: (1) Morphological observation showed that the leaf number of the mutant was 3.3 times as many as that of the wild type, the height of mutant was 2.2 folds that of the wild type, and the mutant had the late-flowering character. (2) Cytological examination showed that the mutant was a normal diploid, and the number of chloroplasts in a guard cell of mutant was 1.3 times that of the wild type. (3) Both chlorophyll a and b content of the mutant were larger than that of the wild type; soluble protein content of the mutant was 1.18 times as much as that of the wild type. Peroxidase isozyme and cytochrome-oxidase isozyme electrophoresis analyses indicated differences between the mutant and the wild type. The soluble protein SDS-PAGE patterns showed the absence of four bands [P1 (114.6 kD), P2 (103 kD), P3 (66.2 kD), P4 (24 kD)] in the mutant. (4) RAPD analysis showed that the similarity index of the mutant and the wild type was 0.612. This suggested that there were changes at DNA level. The mRNA of mutant leaves at flowering stage was isolated; DDRT-PCR was carried out using 10 random primers, using OligodT(15)M (M=A/G/C) as anchor primer. It has been proved that gene expression at anthesis was different between the mutant and the wild type. (5) Genetic observation showed that the mutant was homozygotic and bred true. And the mutant was a late-flowering one.

Chlorophyll↗
PubMed1990

DNA repair and the molecular mechanisms of Bloom's syndrome.

This critical review considers recent work on alterations in DNA repair capacity in Bloom's syndrome as a molecular mechanism for this human disorder. Four main types of DNA repair deficiencies are discussed. These include perturbations in the temporal regulation of DNA repair pathways during the cell cycle, failure to enhance DNA repair pathways during cell proliferation, reduced levels of DNA ligase in Bloom's syndrome cells, and the identification of mutant repair enzyme proteins. These deficiencies are considered in relation to the cellular characteristics of Bloom's syndrome, including delays in DNA replication, hypermutability, and increased incidence of chromosomal aberrations (spontaneously occurring or observed after exposure to environmental agents). The relationship between DNA repair deficiencies and the genetic basis of Bloom's syndrome is described. Previous evidence suggested an autosomal recessive mode of inheritance for Bloom's syndrome. A discussion is presented as to the molecular mechanism through which an alteration in a single gene could result in multiple DNA repair defects.

Base Sequence↗
PubMed1985

[Identification of the number of mutant copies of the genetic transfer factor AP42].

The copy number mutant of genetic transfer factor pAP42 (incFIX) was induced by the treatment of E. coli AP115 cells carrying the plasmid by N-methyl-N-nitro-N-nitrosoguanidine. The copy number mutant pAP42::Tn1cop1 is characterized by the increased copy number. The cells carrying the copy number mutant have a higher ampicillin resistance and higher beta-lactamase activity. Mutant plasmid pAP42: :Tn1cop1 is incompatible with plasmid pAP42 and compatible with plasmids of the other inc-groups of F-like plasmids.

Chromosomes, Bacterial↗
PubMed1994

[Squamous cell carcinoma in oral lichen ruber planus. A clinico-pathological and immunohistochemical study of 11 cases].

The malignant transformation of lichen ruber planus (LRP) is a quite frequent event for which, at present, no clear pathogenetic explanation has been given. Recently, attention has been paid to growth factors and growth factor receptors as possible pathogenetic factors of malignancy, even if not enough studies have been performed to exploit their role in oral diseases. Aim of the present study was to investigate the expression of epidermal growth factor receptor (EGFr) in squamous carcinomas arising in patients with LRP. The study has been performed on formalin-fixed, paraffin-embedded tissue biopsies obtained from 11 patients with previous diagnosis of LRP in which squamous carcinoma had developed. A monoclonal anti-EGFr antibody has been used, with the ABC technique, on histological sections. EGFr has been immunolocalized in basal and suprabasal epithelial layers in LRP with the highest intensity of staining in the areas with strong inflammation. EGFr immunostaining could be detected in dysplastic areas as well, even if the intensity of staining seemed to decrease with severity of dysplasia. In well-differentiated squamous carcinoma EGFr was intensely expressed, especially in mature keratinized cells. Weaker or absent EGFr immunoreaction was detected in moderately and poorly differentiated carcinomas. The results of the present study seem to show that EGFr is expressed in squamous carcinomas arising from LRP. The variability of EGFr expression in inflamed oral mucosa, in dysplastic areas and in tumors with different degrees of differentiation seem to point out that EGFr might somehow regulate the maturation and replication of epithelial cells in LRP and in its malignant counterpart. The speculations achieved from the present study need further evaluation on wider series of cases and, possibly, with different antibodies which would allow the identification of mutant forms of the receptor.

Adult↗
PubMed1994

Identification of temperature-sensitive mutants of the human immunodeficiency virus type 1 protease through saturation mutagenesis. Amino acid side chain requirements for temperature sensitivity.

Human immunodeficiency virus type 1 encodes a protease whose activity is required for the production of infectious virus. An Escherichia coli expression and processing assay system was used to screen 285 protease mutants for temperature-sensitive activity. Fourteen protease mutants had a temperature-sensitive phenotype, and approximately half resulted from conservative amino acid substitutions. Of the 14 substitutions that conferred a temperature-sensitive phenotype, 11 substitutions occurred at 6 positions that represent 3 pairs of residues in the protease that contact each other in the three-dimensional structure. These mutants assist in pinpointing regions of the protease that are important for enzyme activity and stability.

Amino Acids↗
PubMed1993

The role of pharmacogenetics in chemotherapy: modulation of tumour response and host toxicity.

What may appear to be a strategy for investigating pharmacogenetic variation must be seen in the context of an evolving understanding of the molecular biology of drug metabolism. New biochemical and analytical techniques permit the determination of mechanisms of drug action and the molecular biology of host and tumour factors influencing that action. However, the only example of the application of pharmacogenetics in cancer chemotherapy is that of 6MP in the treatment of ALL. This was based on a solid understanding of both the metabolism of the drug and therapeutic response and the ability to measure the polymorphic enzyme in erythrocytes. Identification of the gene responsible for this enzyme is thought to be near, with subsequent identification of mutant forms completing the pharmacogenetic story for this drug. However, pharmacogenetics appears to be an important component in the variability of both toxicity and tumour response for several chemotherapeutic agents. Determination of the complete molecular basis for this variation will no doubt make a major contribution to the increased understanding of host and tumour pharmacology, with the ultimate aim of optimization of therapy.

Antineoplastic Agents↗
PubMed1996

Requirements for dE2F function in proliferating cells and in post-mitotic differentiating cells.

The transcription factor E2F is a target of the retinoblastoma tumor suppressor protein (pRB) and may mediate pRB regulation of S phase entry in mammalian cells. The recent identification of mutant alleles of the Drosophila E2F gene (dE2F) has shown that dE2F is required for embryogenesis. dE2F-mutant embryos lack a co-ordinated program of gene expression which accompanies S phase entry and DNA synthesis declines to levels that are barely detectable. We have investigated the role of the dE2F gene at later stages of development. dE2F is expressed in several larval tissues and is required for cell proliferation in the eye imaginal disc. Surprisingly, dE2F expression persists in post-mitotic cells of the eye disc of third-instar larvae. The loss of dE2F function in these cells causes a novel phenotype, characterized by loss of photoreceptors and abnormal rhabdomere cell morphology. These results show that dE2F is required at multiple stages of development and suggest that E2F may have an important function in post-mitotic cells in addition to its role during cell proliferation.

Animals↗