The expression of proto-oncogenes representative of several functional categories has been investigated during development of mouse male germ cells. The c-raf protooncogene and three members of the ...c-ras gene family were expressed in mitotically active stem cells, throughout the prophase of meiosis and to varying extents in postmeiotic cell types. In contrast, the nuclear proto-oncogenes c-fos, c-jun, and c-myc were specifically expressed at high levels in type B spermatogonia. High levels of c-myc and c-jun RNAs were also detected in spermatocytes early in the prophase of meiosis. The type B spermatogonia represent the last mitotic cell division before entry into meiotic prophase; therefore, these nuclear proto-oncogenes may be involved in altering programs of gene expression at this developmental transition.
ABSTRACT
The Myb protein contacts its recognition sequence by means of direct protein–DNA interactions. We used site-directed mutagenesis in order to substitute amino acids crucial for these contacts ...and probed the mutant proteins for their DNA-binding and transactiving activities. We could show that amino acids involved in direct readout contacts do not contribute equivalently in the recognition process.
Quantitation of nucleic acids by the polymerase chain reaction (PCR) requires coamplification of a control nucleic acid, usually a variant of the sequence to be analyzed, which is added to the sample ...DNA and amplified in competition. Following PCR, amplified sample and control DNAs are separated by electrophoresis and quantitated, for example by measuring the radioactivity incorporated into the products during the PCR. The need for both electrophoretic separation and radioactive labeling has considerably impeded the use of PCR for routine purposes, e.g., in the clinical laboratory, which requires automatic processing of many samples in parallel. We describe here a quantitative PCR procedure which circumvents electrophoretic separation and detection by radioactivity. It uses a point mutated version of the sample DNA as an internal control. After competitive PCR, amplified sample and control DNA are distinguished by an oligodeoxynucleotide ligation assay (OLA) (Landegren et al., Science 241, 1077-1080, 1988) using two oligodeoxynucleotides, one carrying a biotin-group at the 5'-end and another one with either a digoxigenin (specific for sample DNA) or fluorescein moiety (specific for control DNA), respectively, incorporated close to the 3'-end. Biotinylated oligodeoxynucleotides, educts as well as products of the ligation reaction, are immobilized on avidin-coated microtiter plates. Quantitation of digoxigenin and fluorescein-labeled oligodeoxynucleotide ligation products is achieved by an enzyme-linked immunosorbent assay. This method is very well suited for fast automated or semiautomated PCR.
Substrate-assisted catalysis was suggested to be involved in the DNA cleavage reaction of the restriction endonucleases (ENases)
EcoRI and
EcoRV, because experimental evidence exists that the ...phosphate group 3′ to the scissile bond serves to deprotonate the attacking water. Here, we have addressed the question whether this is a general mechanistic feature of the reactions catalyzed by ENases. For this purpose, the cleavage rates of modified and unmodified oligodeoxyribonucleotides (oligos), in which the phosphate group 3' to the scissile bond is substituted by a methyl phosphonate, were measured for 17 enzymes. Only five turned out not to be inhibited by this modification (
BglII,
BstI,
BstYI,
Cfr10I and
MunI); all others cleave the modified substrate at a strongly reduced rate or not at all. By employing a hemisubstituted oligo substrate we were able to further investigate the mechanism of inhibition of the latter group of ENases. Some of them cleave the unmodified strand of the modified substrate with a nearly unaltered rate, whereas the modified strand is cleaved very slowly or not at all (
BamHI,
Bsp143I,
Eco72I,
MflI,
NdeII,
Sau3AI,
XhoII). The others (
AluI,
Cfr9I,
DpnII,
MboI,
PvuII) cleave the modified strand of the modified substrate with a largely reduced rate or not at all. These ENases, however, cleave the unmodified strand with a reduced rate, too. Based on these results we conclude that
BamHI,
Bsp143I,
Cfr9I,
DpnII,
Eco72I,
MboI,
MfII,
NdeII,
PvuII,
Sau3AI and
XhoII may possibly employ substrate assistance in catalysis.
Quantification of alpha 1-fetoprotein (AFP) mRNA in the blood using reverse transcriptase polymerase chain reaction (RT-PCR) could be a useful tool in monitoring the dynamics of minimal residual ...disease in patients with hepatocellular carcinoma (HCC). Since all available assays do not take into account the efficiency of cell separation, RNA extraction and reverse transcription, a competitive RT-PCR assay for quantification of AFP mRNA in relation to the housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH) was established.
Peripheral blood of 22 patients and bone marrow aspirates of 11 patients with hepatocellular carcinoma was monitored perioperatively. Eighteen patients with other hepatic tumours or non-malignant hepatic diseases and 26 healthy blood donors served as controls. Messenger RNA contents were calculated relative to the content of GAPDH mRNA as an indicator of total cell count.
Among HCC patients, 6 of 22 (26%) were positive for AFP mRNA before operation with values ranging from 2 ag/100 fg to 36 ag/100 fg GAPDH mRNA (mean 14). Among bone marrow samples, AFP mRNA was detectable in 5 of 11 (45%) cases, with 4 ag/100 fg to 23 ag/100 fg GAPDH (mean 9). However, AFP mRNA was also detectable in 3 of 18 (17%) control patients and in 2 of 26 (8%) healthy blood donors. Perioperative findings were highly variable.
AFP mRNA is not a specific marker for circulating malignant hepatocytes. Whether definition of a cut-off level or the use of a multimarker-PCR will provide more useful data remains to be established.
The mouse c-mos proto-oncogene RNA is expressed primarily in mouse gonadal tissues and embryos. Until now, the c-mos protein has not been identified. Utilizing two different site-directed affinity ...purified anti-peptide antibodies, we have identified a 43 kDa c-mos protein in mouse testes and in germ cell preparations derived from testes. This 43 kDa testicular protein was found to be structurally related to a bacterially expressed c-mos protein by peptide mapping. Immunoblots of whole mouse sections were employed to establish that the c-mos protein is expressed primarily in the testes.
We have genetically engineered the Arg200---Lys mutant, the Glu144Arg145---GlnLys double mutant, and the Glu144Arg145Arg200---GlnLysLys triple mutant of the EcoRI endonuclease in extension of ...previously published work on site-directed mutagenesis of the EcoRI endonuclease in which Glu144 had been exchanged for Gln and Arg145 for Lys Wolfes et al. (1986) Nucleic Acids Res. 14, 9063. All these mutants carry modifications in the DNA binding site. Mutant EcoRI proteins were purified to homogeneity and characterized by physicochemical techniques. All mutants have a very similar secondary structure composition. However, whereas the Lys200 mutant is not impaired in its capacity to form a dimer, the Gln144Lys145 and Gln144Lys145Lys200 mutants have a very much decreased propensity to form a dimer or tetramer depending on concentration as shown by gel filtration and analytical ultracentrifugation. This finding may explain the results of isoelectric focusing experiments which show that these two mutants have a considerably more basic pI than expected for a protein in which an acidic amino acid was replaced by a neutral one. Furthermore, while wild-type EcoRI and the Lys200 mutant are denatured in an irreversible manner upon heating to 60 degrees C, the thermal denaturation process as shown by circular dichroism spectroscopy is fully reversible with the Gln144Lys145 double mutant and the Gln144Lys145Lys200 triple mutant. All EcoRI endonuclease mutants described here have a residual enzymatic activity with wild-type specificity, since Escherichia coli cells overexpressing the mutant proteins can only survive in the presence of EcoRI methylase. The detailed analysis of the enzymatic activity and specificity of the purified mutant proteins is the subject of the accompanying paper Alves et al. (1989) Biochemistry (following paper in this issue).
