Transcription of bacteriophage lambda DNA in vitro using purine nucleoside 5'-[gamma-S]triphosphates as affinity probes for RNA chain initiation.
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Adenosine Triphosphate↗
Biomedical subjects
A E Reeve
Publications and source records attributed to A E Reeve.
Analysis of RNA initiated in isolated mouse myeloma nuclei using purine nucleoside 5'[gamma-S]triphosphates as affinity probes.
The purine ribonucleoside triphosphate analogues adenosine 5'[gamma-S]triphosphate and guanosine 5'[gamma-T]triphosphate were used as affinity probes for studying RNA chain initiation in isolated nuclei from the mouse myeloma 66.2 cell line. Transcripts initiated with either nucleotide analogue were isolated by affinity chromatography on a mercury-agarose affinity column. The binding was specific and dependent upon the inclusion of the sulfur nucleotide analogues in the in vitro synthetic reaction. Several lines of evidence indicate that the affinity-labeled RNA is initiated in vitro. First, the sulfur nucleotide is recovered in high yield as a single nucleoside 5'[gamma-S]triphosphate 2',3'-monophosphate product following alkaline hydrolysis of RNA bound to the affinity column. Second, authentic ribosomal 5S RNA is known to initiate with GTP; in vitro 5S RNA is bound to mercury-agarose only if [gamma-S]GTP is used as the affinity label in the synthesis, and not if [gamma-S]ATP is used. Under the conditions studied, nuclei incorporated 1.2--2.4 pmole of UMP per 10(6) nuclei per min, and the rate of synthesis was unaffected by substitution of the nucleotide analogues for the normal nucleotides. The percentage of the total RNA synthesized that was incorporated into sequences initiated in vitro was 7.8 +/- 1.5% with [gamma-S]ATP and 9.6 +/- 6.4% with [gamma-S]GTP. The size of the total RNA synthesized, determined by sedimentation on sucrose density gradients containing dimethylsulfoxide, ranged from less than 5S to 45S, and the size of the affinity-labeled sequences ranged from less than 5S to 28S. Approximately half of the incorporation into RNA initiated in vitro was sensitive to a concentration of alpha-amanitin which selectively inhibits polymerase II activity. Most of the remaining incorporation into initiated sequences can be abolished by concentrations of alpha-amanitin that are inhibitory for polymerase III activity. Over 70% of the total incorporation into ribosomal 5S RNA transcripts was into sequences initiated in vitro. This initiation was catalyzed by polymerase III and was specific for GTP as the initiating nucleotide. The RNAase T1 fingerprint of the newly initiated 5S RNA indicates that this gene is accurately initiated and faithfully elongated in vitro. The use of these affinity label probes provides much greater sensitivity for studying the initiation of RNA in vitro.
Amanitins↗
Studies on gene transcription in vitro by analysis of the primary transcripts.
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Cell Nucleus↗
Incorporation of purine nucleoside 5'-[gamma-S]triphosphates as affinity probes for initiation of RNA synthesis in vitro.
Synthetic DNA templates were transcribed by Escherichia coli RNA polymerase using nucleoside 5'-[gamma-S]triphosphates as one of the nucleotide substrates. Substitution of the thiol analogues for the normal nucleotides had no effect on the rate of RNA synthesis. RNA synthesized with either adenosine 5'-[gamma-S]triphosphate or guanosine 5'-[gamma-S]triphosphate was isolated with high efficiency on mercury-agarose columns prepared by activation with low concentrations of cyanogen bromide. Sulfur was shown to be incorporated at the 5' end of RNA by identification of the tetraphosphate HSpppA32p liberated after alkaline hydrolysis of HS(A-32pU)n (alternating copolymer synthesized by the action of E. coli RNA polymerase on d(A-T)n-d(A-T)n with adenosine 5'-[gamma-S]triphosphate and uridine 5'-[alpha-32P]triphosphate as substrates). Transcripts elongated but not initiated with these thiol analogues did not bind to the affinity column. This technique provides an extremely sensitive assay for RNA synthesis initiation in vitro, since initiated transcripts containing radiolabel throught the entire transcript can be isolated.
Chromatography, Affinity↗
Loss of a Harvey ras allele in sporadic Wilms' tumour.
Genomic changes within chromosome band 11p13 appear to have a role in the initiation of Wilms' tumour. The human Harvey ras oncogene, c-Ha-ras 1, has been located by Jhanwar et al. immediately adjacent to this region at band 11p14 .1, although several groups have assigned the gene more distally at band 11p15 . We have examined tumour DNA from two cases of sporadic Wilms' tumour, and report here that in both cases one of the two constitutional c-Ha-ras 1 alleles was absent. One tumour had a reciprocal translocation between the short arm of chromosome 11 (at band 11p13), and the long arm of chromosome 12, with no visible loss of chromosomal material. The loss of a c-Ha-ras 1 allele in association with this translocation indicates that a submicroscopic deletion had occurred. The resulting hemizygosity may have had a role in tumour initiation. Our results indicate that the c-Ha-ras 1 gene and the 'Wilms' tumour locus' may be in close proximity. It would, therefore, be premature to exclude the possibility that these two sites are functionally related.
Alleles↗
Expression of insulin-like growth factor-II transcripts in Wilms' tumour.
Wilms' tumour probably arises from embryonal kidney cells and occurs in both hereditary and sporadic forms. Knudson and Strong have suggested that both forms of the disease are initiated by two mutational events. In the case of the inherited form, cytogenetic evidence indicates that a germline deletion of chromosome band 11p13 may correspond to one of the two mutations. DNA mapping evidence is consistent with the notion that the tumour susceptibility gene (Wg) on chromosome 11 is actually recessive. Comings has proposed that the dominantly inherited tumours may arise by the inactivation or loss of a diploid pair of regulatory genes which normally suppress the expression of a structural transforming gene (Tg). It has recently been suggested that the N-myc oncogene may serve as a transforming gene in retinoblastoma, although no such gene has yet been identified in Wilms' tumour. We now report that in four cases of Wilms' tumour, insulin-like growth factor-II (IGF-II) transcripts are highly elevated compared with the adjacent normal kidney. In addition, we have mapped the gene for IGF-II to chromosome band 11p14.1, which is in the immediate vicinity of Wg. These findings suggest that IGF-II may be involved in the aetiology of Wilms' tumour.
Cell Cycle↗
Genomic diversity correlates with clinical variation in Ph'-negative chronic myeloid leukaemia.
The Philadelphia chromosome (Ph') is found in the blood cells of about 90% of patients with chronic myeloid leukaemia (CML) and usually results from the reciprocal chromosome translocation t(9;22). This translocation relocates the proto-oncogene c-abl, normally found on chromosome 9q34, to within the breakpoint cluster region (bcr) on chromosome 22q11 (refs 3-8). The juxtaposition of c-abl and the 5' portion of bcr appears to be the critical genomic event in CML and results in a novel 8-kilobase (kb) fused abl/bcr transcript and a c-abl-related protein of relative molecular mass 210,000 (ref.11). About 10% of adult patients diagnosed as CML lack the Ph' chromosome; they represent a heterogeneous group of disorders which are difficult to diagnose precisely. We have examined five patients with CML whose leukaemic cells have a normal karyotype. We report here that two of the patients showed the same genomic change as occurs in Ph'-positive CML, but the change resulted from a mechanism other than chromosomal translocation. The remaining three patients showed no genomic rearrangement. This genomic diversity correlated with the clinical differences between the patients.
Adult↗
Good prognosis of cellular mesoblastic nephroma with hyperdiploidy and relaxation of imprinting of the maternal IGF2 gene.
A large congenital mesoblastic nephroma (CMN) of combined classical and and cellular histological structure was removed from a 1-month-old female infant. The tumor extended extrarenally and may have been incompletely excised. Tumor tissue showed a mosaic hyperdiploidy with 54 chromosomes in the hyperdiploid line. No other antitumor therapy was given and there has been no recurrence after 4 years. Genomic imprinting normally prevents transcription of the maternal gene for insulin-like growth factor 2 (IGF2). Relaxation of IGF2 imprinting leading to abnormal transcription of the maternal gene is found in a majority of Wilms' tumors and in other malignant neoplasms. The biallelic transcription of IGF2 demonstrated in the CMN from this case is consistent with abnormal transcription of the maternal allele. Relaxation of imprinting of the maternal IGF2 gene or abnormal expression of the gene through other mechanisms may have a role in the genesis of CMN or the cellular subtype.
Diploidy↗