Studying the Chemical Reactivity Properties of Cytosine and its Antiviral Analogues: Zebularine, 5-Aza-Cytosine And 5-Aza-5,6-Dihydro-Cytosine through Density Functional Theory

Zebularine, 5-aza-cytosine and 5-aza-5,6-dihydro-cytosine are structurally similar to cytosine, but their biological functions are rather different. Cytosine can be methylated which is a gene lesion that can cause human disease. On the contrary, zebularine and 5-aza-cytosine are inhibitors of DNA methylation. 5-aza-5,6-dihydro-cytosine is specifically designed to induce lethal mutagenesis in HIV for its structurally variability. Here, theoretical research into their chemical properties through density functional theory is reported. Molecular hardness and molecular electronic surface potential were analysed. Compared to cytosine, the main reason for the inability of methyl addition of zebularine is the reduced nucleophilicity of C5 atom. The lack of a hydrogen atom at N5 atom in 5-aza-cytosine is responsible for the incomplete reaction of methyl transfer. Variability of 5-aza-5,6-dihydro-cytosine is responsible for the mutagenesis treatment by paring with guanine or adenine with its different tautomers. Aspect of these chemical reactivities can be accounted for the distinctive biological functions of these molecules.

Partial gene deletion in a family with factor X deficiency

The presence of gene lesions in coagulation factor X (FX, Stuart factor) was investigated in patients with FX deficiency or an FX abnormality (FX Friuli). The proposita had a heterozygous partial deletion of the FX gene with severe deficiency of FX activity and antigen. The lesion, which was inherited from her mother, removes the 3′ portion of the gene coding for the catalytic domain of the factor. In this family, two differently affected FX genes are present, leading to double heterozygosity of the proposita and thus excluding consanguinity of parents. An apparently normal gene structure was observed in the other patient with FX abnormality, suggesting the presence of a small gene lesion.

Partial gene deletion in a family with factor X deficiency

The presence of gene lesions in coagulation factor X (FX, Stuart factor) was investigated in patients with FX deficiency or an FX abnormality (FX Friuli). The proposita had a heterozygous partial deletion of the FX gene with severe deficiency of FX activity and antigen. The lesion, which was inherited from her mother, removes the 3′ portion of the gene coding for the catalytic domain of the factor. In this family, two differently affected FX genes are present, leading to double heterozygosity of the proposita and thus excluding consanguinity of parents. An apparently normal gene structure was observed in the other patient with FX abnormality, suggesting the presence of a small gene lesion.

A POINT MUTATION AND A GENE DELETION OF FVIII GENE IN SEVERE HAEMOPHILIA

The presence of Factor VIII (FVIII) gene lesions has been investigated in 100 haemophilia A patients using cDNA probes for the 3'part of FVIII gene (exons 14-26 ).In two related severe patients without inhibitor a deletion removesthe exon 26; the gene lesion has been confirmed with several restriction enzymes and has been shown by densitometry of the autoradiographic pattern in a woman of the same family. The complete deletionof the exon 26 has been described by Gitschier et al. in a patient with inhibitor. Thus the comparison of the end points of the two deletions could help to define the mechanism originating these gene lesions and the relation between gene lesions and the presence of antibody.In a patient with severe Haemophilia and without inhibitor a mutation removing the TaqI site in the exon 24 and originating an abnormal band of 4.2 Kb has been found. A C→T transition in this TaqI site, originating a nonsense codon and a new Hindlll site, has been reported by Gitschier et al in a patient presenting inhibitor. The DNA from our patient tested with Hindlll shows a normal pattern thus indicating a C→T transition in the antisense strand. This mutation should causean aminoacid change (CGA→CAA, Arg→Gln) possiblyresponsible for the FVIII inactivation but that does not remove theantigenic determinants present in the COOH terminal part of FVIII.In addition the same mutation has been observed in an unrelated (asdemonstrated by RFLPs analysis) Italian haemophilic patient confirming the observation of Youssoufian et al that TaqI sites are mutational hot spots in FVIII gene.

A FREQUENT TAQI RFLP AND A GENE LESION OR RARE TAQI RFLP IN THE VON WILLEBRAND FACTOR GENE

A cDNA for vonWi11ebrand factor (vWf) has been used to investigate lesions and RFLPs in the vWf gene.In hybridizations with the 3'cDNA portion (a 2 Kb SacI fragment) a frequent polymorphism has been found with TaqI restriction enzyme. The alleles are 3.3 Kb and 2.6 Kb ; the frequencies of 0.51 and 0.49 respectively enable to investigate an appreciable portion of vW disease (vWd) families.In a patient with type III vWd an abnormal TaqI pattern has been observed. A 4.5 Kb band is absent and an additional band of 2.3 Kb is present.This pattern has been inherited from the consanguineous heterozygous parents and has been traced in several members of this large family. The presence of the abnormal gene pattern is related to total or partial vWf deficiency in the family and has not been found in several normal subjects. The BamHI and BglII restriction patterns are normal and suggest a small mutation originating a new TaqI site.These findings are compatible with a gene lesion or a rare RFLP.Work supported by Ricerca Sanitaria Finalizzata Regione Emilia Romagna.

Revertants of an S49 cell mutant that expresses altered cyclic AMP-dependent protein kinase

Dibutyryl adenosine 3',5'-phosphate (Bt2cAMP)-sensitive (Bt2cAMPS) revertants were isolated from a resistant S49 cell mutant carrying a structural gene lesion in the regulatory subunit of cAMP-dependent protein kinase (cA-PK). This was accomplished with a counter-selection in which, first, Bt2cAMP was used to reversibly arrest revertants, and then a sequence of treatments with bromodeoxyuridine, 33258 Hoechst dye, and white light was used to kill cycling mutant cells. Reversion rates in nonmutagenized cultures could not be accurately measured, but spontaneous revertants do occur and with frequencies of less than 10(-7) to 10(-5). The mutagens ethyl methane sulfonate (EMS), N-methyl-N'-nitro-N-nitro-soguanidine (MNNG), and ICR191 increased the reversion frequency. In all cases, reversion to Bt2cAMP sensitivity was associated with restoration of wild-type levels and apparent activation constant for cAMP of cA-PK. MNNG induced revertants whose cell extracts contained cA-PK activity distinguishable from that of wild type by thermal liability. EMS did not. The counter-selection effectively isolates rare phenotypes and is therefore a useful tool in further somatic genetic experiments. The association of reversion with alterations in cA-PK function supports all previous data from this and other laboratories implicating cA-PK as the intracellular mediator of cAMP effects. Reversion is probably the result of a mutational event. Induction of reversion by ICR191 suggests the existence of a novel mechanism for generating revertants in somatic cells.