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Biomedical subjects

A J Gordon

Publications and source records attributed to A J Gordon.

At least 19 recordsLinked to original sources
PubMed1995

Isolation of an ubiquitously expressed cDNA encoding human dynamin II, a member of the large GTP-binding protein family.

Dynamin (Dyn) is a member of a novel group of GTPases which was initially identified as a microtubule-binding protein with a role in vectorial movement. Three distinct Dyn-encoding genes (DYN I, II and III), with a neuronal-, ubiquitous or testis-specific expression, respectively, have been identified in rat. In man, only DYN I has so far been characterized. We have previously isolated a genomic DNA fragment implicated in the correction of mitomycin C hypersensitivity of cells from a Fanconi anemia patient belonging to genetic complementation group D (FA(D)). Using this probe, we have cloned a human complementary DNA designated hDYN II encoding a ubiquitous Dyn isoform. The predicted protein consists of 866 amino acids (97.5 kDa). Dyn proteins exhibit a high degree of evolutionary conservation: hDyn II is 98% identical to rat Dyn II and 73% identical to hDyn I. A unique 3.6-kb transcript is found in all human tissues examined and it is more abundant in skeletal muscle and heart. This transcript is also expressed in tissue-culture cells. The hDYN II message is present and not mutated in the FA(D) patient studied. In addition to the GTP-binding domain and motifs associated with regulatory function, the hDyn II protein contains a noticeable number of concensus motifs for p34Cdc2 kinase phosphorylation which may indicate a potential role at the G2/mitosis transition. The sequence reported here should allow a more complete analysis of Dyn function(s) in man.

Amino Acid Sequence↗
PubMed1995

Inversions with deletions and duplications.

Complex mutational events, including de novo inversion with deletion and duplication of sequence, have been observed but are difficult to model. We propose that nascent leading-strand misalignment upon the lagging-strand template during DNA replication can result in the inversion of sequence. The positioning of this misalignment and of the realignment of the leading strand back into the leading-strand template will determine if the inversion is accompanied by deletion and duplication of sequence. We suggest that such strand misalignment-realignment events may occur at the replication fork during concurrent DNA replication.

Base Sequence↗
PubMed1992

Influences on biomedicine in rural Dominican Republic: an analysis of process.

This paper, based on fieldwork among agrarian reform beneficiaries in the Dominican Republic, examines the utilization of health care. Specific attention is given to the consumption of private medical care and its relationship to changes in the system of land tenure, policy in the agrarian reform, and the roles of physicians. A principal concern of the paper is the examination of the adaptive strategies of beneficiaries of the reform in light of political and economic influences. A second concern is the consideration of the integration of critical medical anthropology's perspectives on the political economy with a perspective on decision-making and adaptive strategies.

Agriculture↗
PubMed1992

The influence of local DNA sequence and DNA repair background on the mutational specificity of 1-nitroso-8-nitropyrene in Escherichia coli: inferences for mutagenic mechanisms.

We have examined the mutational specificity of 1-nitroso-8-nitropyrene (1,8-NONP), an activated metabolite of the carcinogen 1,8-dinitropyrene, in the lacI gene of Escherichia coli strains which differ with respect to nucleotide excision repair (+/- delta uvrB) and MucA/B-mediated error-prone translesion synthesis (+/- pKM101). Several different classes of mutation were recovered, of which frameshifts, base substitutions, and deletions were clearly induced by 1,8-NONP treatment. The high proportion of point mutations (> 92%) which occurred at G.C sites correlates with the percentage of 1,8-NONP-DNA adducts which occur at the C(8) position of guanine. The most prominent frameshift mutations were -(G.C) events, which were induced by 1,8-NONP treatment in all strains, occurred preferentially in runs of guanine residues, and whose frequency increased markedly with the length of the reiterated sequence. Of the base substitution mutations G.C-->T.A transversions were induced to the greatest extent by 1,8-NONP. The distribution of the G.C-->T.A transversions was not influenced by the nature of flanking bases, nor was there a strand preference for these events. The presence of plasmid pKM101 specifically increased the frequency of G.C-->T.A transversions by a factor of 30-60. In contrast, the -(G.C) frameshift mutation frequency was increased only 2-4-fold in strains harboring pKM101 as compared to strains lacking this plasmid. There was, however, a marked influence of pKM101 on the strand specificity of frameshift mutation; a preference was observed for -G events on the transcribed strand. The ability of the bacteria to carry out nucleotide excision repair had a strong effect on the frequency of all classes of mutation but did not significantly influence either the overall distribution of mutational classes or the strand specificity of G.C-->T.A transversions and -(G.C) frameshifts. Deletion mutations were induced in the delta uvr, pKM101 strain. The endpoints of the majority of the deletion mutations were G.C rich and contained regions of considerable homology. The specificity of 1,8-NONP-induced mutation suggests that DNA containing 1,8-NONP adducts can be processed through different mutational pathways depending on the DNA sequence context of the adduct and the DNA repair background of the cell.

Base Sequence↗
PubMed1992

Resolution and characterization of polymorphic DNA by SSCP and chemical cleavage methodologies.

A variety of techniques have been developed to detect single-base changes for the two different purposes. One is the detection of mutational events without phenotypic selection, and another is the rapid and conventional identification of mutations such as the specific base changes related to activation of oncogene, genetic diseases, etc. In this study, the utility of the two methods, single strand conformation polymorphism (SSCP) and chemical cleavage, was explored using 13 E. coli lacI- mutations cloned onto M13 phase. The 167 base region encompassing mutations was amplified by PCR as dsDNA. Following denaturation, these PCR products were analyzed by non-denaturing polyacrylamide gel electrophoresis (SSCP) and the separation of the ssDNA fragment carrying the altered sequence from the original sequence was found to be dependent on the location and type of the change. Hetero duplexes of changed/original sequences were also prepared by hybridization of the above PCR products. Mismatched C and T bases were modified by hydroxylamine and osmium tetroxide, respectively, and subsequently treated with piperidine to analyze the cleaved DNA fragments on a polyacrylamide gel (Chemical Cleavage). The cleavage efficiency was also found to be influenced by the type of mismatch and its surrounding sequence. Such observed characteristics should contribute to a better appreciation for these types of mutational systems, which in turn should lead to insight into the mechanisms of mutagenesis.

Base Sequence↗
PubMed1991

Spontaneous and 9-aminoacridine-induced frameshift mutagenesis: second-site frameshift mutation within the N-terminal region of the lacI gene of Escherichia coli.

A novel forward mutational system, based on the acquisition of an Iq-d dominant phenotype from an initial Iq- recessive state, was used to identify second-site frameshift mutation [+/- 1(+/- 3n) events] within the N-terminal region of the lacI gene of Escherichia coli. The DNA sequences are described of forty-six spontaneous and twenty 9-aminoacridine(9-AA)-induced second site mutations. Although -1 frameshift events dominate both spectra, the nature and site specificity of these events clearly distinguish two mutational distributions. The spontaneous distribution contains two -(A:T) frameshift hotspots; one within a monotonic A5 run (9 occurrences), the other at a 5'-CACAACAAC-3' sequence (12 occurrences). In contrast 17 of the 20 mutations recovered after 9-AA treatment involve the loss of a G:C pair, 14 of which occur at a single site (5'-CGGGC-3'). The striking specificity of the observed mutational hotspots is of interest since this open genetic target contains similar sequences which were infrequently recovered.

Aminacrine↗
PubMed1991

Social networks and recovery: one year after inpatient treatment.

In an effort to understand the effect of social networks on outcome for treatment of alcoholism, a 1-year follow-up prospective study of 156 inpatients was undertaken. Treatment outcome was associated with diverse aspects of the social network: whether individuals were family, friends, or co-workers; the type of relationship--whether or not drinking together was part of social interaction; and the presence of social support, both perceived support and support through participation in treatment. Hierarchical path analysis using Lisrel 6 was used to analyze the data. The active support of co-workers not regularly drinking with the patient and the perceived support of co-workers without respect to their drinking were influential in recovery, exerting positive and negative effects, respectively. The indirect effect of other variables indicates that nondrinking and drinking family and friend relationships also exert positive and negative effects, respectively. The authors suggest that a major factor in recovery is the ability to elicit and receive support. Improving this ability may be a fruitful objective in treatment.

Adolescent↗
PubMed1991

Complex choristoma of the eyelid containing ectopic cilia and lacrimal gland.

A 2-year-old girl was born with an aggregate of ectopic cilia in the right upper eyelid that intermittently produced tears. During elective excision, the cilia were found to be clustered tightly with prominent bulb-like follicles. A separate lobe of lacrimal gland tissue was adjacent to the roots of the cilia just posterior to the orbital septum with no direct communication with the lacrimal gland. Histopathologically, the lesion contained multiple large hair follicles in the dermis with accompanying adnexal structures. Large ectopic lobules of lacrimal gland tissue also were present. Postoperatively, the child had a good cosmetic result and has remained asymptomatic over an 18-month follow-up period. To our knowledge, this is the first reported case of an eyelid choristoma containing ectopic cilia and lacrimal gland. Ectopic cilia alone is a very rare anomaly of the lashes, with only nine cases reported in the literature.

Child, Preschool↗
PubMed1991

The mutational specificity of 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF2) in the lacI gene of Escherichia coli.

We have determined the mutational specificity of the 5-nitrofuran derivative furylfuramide (AF2) in the lacI gene of Escherichia coli. Treatment of a delta uvrB, pKM101 strain with 1 M AF2 yielded a mutation frequency approximately 300 times greater than that of untreated controls. Of the 165 AF2-induced mutants analysed by DNA sequencing, 145 were base substitution mutations, 11 were frameshifts, and the remainder small deletions, tandem base substitutions and complex mutations. Base substitution occurred primarily (greater than 93%) at G:C base pairs. The proportions of the various mutations are very similar to those that have been reported for AP sites. We suggest that the principal mechanism for AF2 mutagenesis is the formation of an adduct which depurinates to yield AP sites that serve as a substrate for error-prone repair. Seventy-two of the mutations occurred at four 5'-TGC-3' sites. The majority (10/11) of the frameshift mutations occurred at one such hotspot and could have been templated by an inverted repeat less than 100 bp removed from the site of the mutation.

Base Sequence↗
PubMed1991

The action of 1-nitroso-8-nitropyrene in Escherichia coli: DNA adduct formation and mutational consequences in the absence of nucleotide excision-repair.

To study the mechanisms of mutagenesis by the carcinogen 1,8-dinitropyrene we have determined the DNA adducts formed and mutations induced by its partially activated metabolite 1-nitroso-8-nitropyrene (1,8-NONP) in an Escherichia coli strain deficient in nucleotide excision-repair. Using DNA sequence analysis we have characterized a collection of 159 lacI- mutations recovered following treatment with 1,8-NONP. The mutational spectrum was dominated by -1 frameshifts (110 events) in runs of contiguous G or C residues. Frameshift frequency was observed to increase with the length of the reiterated sequence. Two mutations involved the loss of GpC from alternating (GpC)n sequences. The ratio of -1:-2 events observed following 1,8-NONP treatment was markedly different from that induced by N-acetoxy-N-acetyl-2-aminofluorene in the same genetic target. Other mutational classes recovered included 'spontaneous' hotspot mutations (19 events), base substitutions (12 events), deletions (7 events), one duplication, one + (A:T) frameshift and one mutation containing closely juxtaposed -(G:C) events. Of the 125 point mutations characterized, 124 occurred at G:C sites. The site specificity of mutation was consistent with the 32P-postlabeling profile of 1,8-NONP-DNA adducts which showed that 95% of the adducts migrated to the same position on the TLC plates as the guanine C(8) adduct 1-N-(2'-deoxyguanosin-8-yl)-amino-8-nitropyrene. Two minor 1,8-NONP-DNA mutations were also detected, one at dG/dC sites, and the other at dA/dT sites.

Animals↗
PubMed1990

Mutational specificity of alkylating agents and the influence of DNA repair.

Alkylating treatments predominantly induce G: C = greater than A:T transitions, consistent with the predicted significance of the miscoding potential of the O6-alG lesion. However, the frequency and distribution of these events induced by any one compound may be diagnostic. SN1 agents that act via an alkyldiazonium cation, such as the N-nitroso compounds, preferentially generate G: C = greater than A:T transitions at 5'-RG-3' sites, while the more SN2 alkylsulfates and alkylalkane-sulfonates do not. The precise nature of this site bias and the possibility of strand bias are target dependent. The extent of this site bias and the contribution of other base substitutions are substituent size dependent. A similar 5'-RT-3' effect is seen for A:T = greater than G:C transitions, presumably directed by O4-alT lesions. The 5'-RG-3' effect, at least, likely reflects a deposition specificity arising from some aspect of helix geometry, although it may be further exaggerated by alkylation-specific repair. Excision repair appears to preferentially reduce the occurrence of ethylation-induced G:C = greater than A:T and A:T = greater than G:C transitions at sites flanked by A:T base pairs. This may be due to an enhancement of the helical distortion imposed by damage at such positions. A similar effect is not seen for methylation-induced mutations and in the case of propyl adducts, the influence of excision repair on the ultimate distribution of mutation cannot be as easily defined with respect to neighbouring sequence.

Alkylating Agents↗
PubMed1990

Non-phenotypic selection of N-methyl-N'-nitro-N-nitrosoguanidine-directed mutation at a predicted hotspot site.

The striking mutational specificity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) exhibited in the lacI gene in Escherichia coli allows comment on the phenotypic consequences of mutation at specific sequences that are not recovered after MNNG mutagenesis. We predict that the I+ phenotype is maintained when such silent positions are substituted by amino acids whose codons are generated by the MNNG-directed G:C----A:T transition. We chose the mutationally silent Gly200 codon (an MNNG hotspot motif sequence) to test this prediction. Through MNNG mutagenesis we have generated, identified and isolated a G:C----A:T transition at position 627 (5'-G-G-C-3') under non-selective conditions which creates the Gly200----Asp substitution. The I+ phenotype is retained for this altered repressor.

Bacterial Proteins↗
PubMed1990

Induction of specific frameshift and base substitution events by benzo[a]pyrene diol epoxide in excision-repair-deficient Escherichia coli.

We have determined the DNA alterations recovered after treatment with (+-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+-)-anti-BPDE] in the lacI gene of excision-repair-deficient (Uvr-) Escherichia coli. The high induction of -(G:C) frameshifts, G:C----T:A and A:T----T:A transversions, and the presence of complex mutations of a particular motif, distinguish the mutational distribution recovered in the Uvr- strain, although other mutational classes, including -(A:T) frameshifts and duplications, were also moderately induced. The great majority of -(G:C) frameshifts, the predominant mutation recovered, occurred in runs of G residues. The G:C----T:A transversion was found to occur more frequently (10/12 occurrences) at 5'-Y-G-3' sites, sequences at which the labile BPDE:N7G adduct has been predicted to occur. Moreover, the relative proportions of G:C----T:A, A:T----T:A and G:C----A:T mutations correlate well with the expected proportions of alkali-labile lesions at G, A and C residues. These results support the model that (+-)-anti-BPDE-induced base substitution mutagenesis in E.coli proceeds through abasic intermediates across from which adenine is preferentially incorporated during replication.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1988

Influence of neighboring base sequence on the distribution and repair of N-ethyl-N-nitrosourea-induced lesions in Escherichia coli.

N-Ethyl-N-nitrosourea-induced mutations occurring within a 180-base pair target in the lacI gene of Escherichia coli were characterized by DNA sequencing. In total, 109 mutations were characterized in a wild-type background and 100 in an excision-repair-deficient (UvrB-) background. The majority of mutations induced in the two backgrounds (77 and 85%, respectively) were G:C = greater than A:T transitions, presumably resulting from miscoding O6-ethylguanine lesions. A significant proportion of the mutations (17 and 15%, respectively) were A:T = greater than G:C transitions, which probably result from miscoding O4-ethylthymine lesions. An analysis of the distribution of both types of mutation in the two backgrounds reveals two distinct influences of neighboring base sequence. These effects apply equally to both the G:C = greater than A:T and A:T = greater than G:C transitions. Firstly, miscoding lesions are most likely to occur at 5'-purine-G-3' or 5'-purine-T-3' sites. Secondly, the excision-repair machinery is less efficient at removing both O6-ethylguanine and O4-ethylthymine lesions which are flanked on both sides by G:C base pairs. Thus, in the wild-type spectrum an overabundance of transitions occurs at a 5'-G-G-G/C-3' or 5'-G-T-G/C-3' sequence (where the mutated base is underlined).

Base Sequence↗
PubMed1988

Missense mutation in the lacI gene of Escherichia coli. Inferences on the structure of the repressor protein.

The lac repressor has been studied extensively but a precise three-dimensional structure remains unknown. Studies using mutational data can complement other information and provide insight into protein structure. We have been using the lacI gene-repressor protein system to study the mutational specificity of spontaneous and induced mutation. The sequencing of over 6000 lacI- mutations has revealed 193 missense mutations generating 189 amino acid replacements at 102 different sites within the lac repressor. Replacement sites are not distributed evenly throughout the protein, but are clustered in defined regions. Almost 40% of all sites and over one-half of all substitutions found occur within the amino-terminal 59 amino acid residues, which constitute the DNA-binding domain. The core domain (residues 60 to 360) is less sensitive to amino acid replacement. Here, substitution is found in regions involved in subunit aggregation and at sites surrounding residues that are implicated in sugar-binding. The distribution and nature of missense mutational sites directs attention to particular amino acid residues and residue stretches.

Amino Acid Sequence↗
PubMed1988

N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation in a RecA strain of Escherichia coli.

274 N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced forward mutations in the lacI gene of an Escherichia coli RecA- strain were cloned and sequenced. Base substitutions accounted for 264 mutations and consisted of 261 G:C----A:T transitions (including one double mutant with two G:C----A:T transitions separated by 25 base pairs), two A:T----G:C transitions and one A:T----T:A transversion. Therefore, 263 of the 274 mutations (all the transitions) can be explained as a result of the direct mispairing of O6-methylguanine, and O4-methylthymine residues during DNA synthesis. The source of the transversion is not known. The remaining mutations, one 16-base pair deletion, two -1 frameshifts and 7 frameshifts at the lacI frameshift hotspot, are located in runs of identical bases or flanked by directly repeated DNA sequences and can therefore be explained by template slippage events during DNA synthesis. The observed distribution of mutations recovered is identical to that found in a RecA+ background indicating little involvement of RecA function in MNNG-induced mutation. Analysis of neighbouring base sequence revealed that the G:C----A:T transition was 6 times more likely to be recovered if the mutated guanine residue was preceded by a purine rather than a pyrimidine. A most striking aspect of this distribution concerns particular residues in the core domain of the lac repressor protein. Within this domain the great majority of mutations generate nonsense codons or alter Gly codons.

Base Composition↗
PubMed1988

Mutational specificity of N-methyl-N-nitrosourea in the lacI gene of Escherichia coli.

The mutational specificity of the carcinogenic alkylating agent N-methyl-N-nitrosourea (MNU) was investigated through the DNA sequence characterisation of 104 lacI-d mutations induced by this agent in the lacI gene of Escherichia coli. The vast majority (95%) of MNU-induced mutations were G:C----A:T transitions. An analysis of neighbouring base sequence revealed that this transition was almost 10 times more likely to occur if the mutated guanine residue was preceded (5') by a purine (R) rather than a pyrimidine (Y); apart from this 5'-R-G-3' influence no other site specificity of mutation was observed. This 5'-flanking base influence on mutability has also been observed in this system with other mechanistically similar alkylating agents.

Base Sequence↗