A fidelity assay using "dideoxy" DNA sequencing: a measurement of sequence dependence and frequency of forming 5-bromouracil X guanine base mispairs.

DNA replication fidelity has been assayed by using a modified DNA sequencing reaction. In one experimental approach, dideoxycytidine 5'-triphosphate (ddCTP) was used as a chain terminator during replication of M13 phage DNA by the large fragment of DNA polymerase I. The deoxyribonucleotide analogue BrdUTP was used to compete against ddCTP-induced chain terminations as an assay for B X G base mispairing (B represents bromodeoxyuridine when the analogue is present as a base pair or base mispair). By comparing BrdUTP to dCTP for competition against ddCTP, an average misincorporation frequency for BrdUMP of 0.2% was found. A similar average misincorporation frequency has been measured previously for the incorporation of radioactively labeled BrdUMP and dCMP into the synthetic template-primer poly-[d(G,T)] X oligo(dA). The advantage of the sequencing method is that an error frequency is determined for each template guanine in a defined DNA sequence, thus providing information on the effect of neighboring base sequences on fidelity. Misincorporation frequencies varied no more than 5-fold among 50 template guanines tested. The approach used here is not limited for use with nucleotide analogues but is generally applicable in determining misincorporation frequencies and sequence specificities for any deoxynucleoside triphosphate substrate. In a second experimental approach, base mispairing between bromouracil and guanine was demonstrated directly by using 5-bromodideoxyuridine 5'-triphosphate (BrddUTP). A comparison of chain terminations attributable to BrddUTP and to dideoxythymidine 5'-triphosphate (ddTTP) revealed that B X A and T X A base pairs formed at about the same rate, whereas B X G mispairs occurred 4-10 times more frequently than T X G. The elevation in the frequency of B X G over T X G mispairs is consistent with the mutagenic behavior of the base analogue.

Heritable fragile sites on human chromosomes. VII. Children homozygous for the BrdU-requiring fra(10)(q25) are phenotypically normal.

A brother and sister have been detected who are homozygous for the bromodeoxyuridine (BrdU)-requiring fragile site at 10q25. The children are phenotypically normal, indicating that homozygosity for this fragile site is harmless, at least during childhood.

[Frequency of aberrations in uni- and biliary 5-bromodeoxyuridine-substituted chromatids]. / Chastota aberratsii v odno- i dvu-5-bromdezoksiuridin-zameshchennykh khromatidakh.

Chinese hamster cells and human lymphocytes were incubated with BrdUrd during two cycles of replication and stained using the "harlequine" technique. The G2 cells were irradiated with 1 Gy of gamma-rays from the 60 Co source. The frequency of breaks in light (i.e. bifiliary substituted) chromatids was significantly lower, as compared with dark (i.e. unifiliary substituted) ones. The comparison of these data with the previously published suggests that the relative frequency of breaks in uni- and bifiliary substituted chromatids depends on the energy of quanta. A hypothesis is discussed that a resonance absorption of energy by Br atoms and an increased interaction between BrdUrd-substituted structures, which favours the repair of double-strand breaks, can explain the noted regularity.

Cartilage cell differentiation: review.

Differentiation of cartilage cells from embryonic precursor cells is characterized by the onset of biosynthesis of at least two cartilage-specific gene products, type II collagen and cartilage-specific chondroitin sulfate proteoglycan (CSPG). Biochemical and immunological assays for these compounds now allow rapid, quantitative, and specific determination of the onset of cartilage differentiation, and present several advantages over assays that use histochemical stains or [35S]-sulfate incorporation into glycosaminoglycans. Chondrogenic differentiation also is associated with the formation of extracellular, high MW proteoglycan (CSPG) aggregates containing hyaluronic acid and the loss of fibronectin, or LETS protein, a cell surface glycoprotein found on presumptive chondroblasts, fibroblasts, and several other cell types. Comparatively little insight has been gained recently regarding the mechanism of cartilage cell differentiation. A number of factors or "inducers" of cartilage differentiation, such as chondroitin sulfate proteoglycan, notochord, spinal cord, low oxygen tension, and collagen substrates, increase the amount of glycosaminoglycan synthesis per cell, but the question remains open as to whether these factors also selectively increase the number of cells differentiating from precursor cells into chondroblasts, or whether they only increase cell viability. Other factors, such as conditioned medium from chondrocyte cultures, increase significantly the number of chondrocyte colonies arising in mass cultures of limb bud mesenchyme, but differentiation of nonchondrogenic cells is stimulated as well. Similarly, many inhibitors of cartilage differentiation, such as BrdUrd and 6-amino nicotinamide, also inhibit myogenic differentiation. It is possible that a unique and specific inducer or regulating factor of cartilage cell differentiation may not exist, for cartilage differentiation of normal embryonic mesenchyme can be triggered by a variety of environmental conditions, such as cell density, pH, potassium ion concentration, and fetal calf serum. These results imply that the temporal and spatial controls of cartilage differentiation are governed by environmental influences that are each of rather low specificity, but which together synergistically generate a morphogenetic control of high specificity. Signals which appear able to mimic those controlling normal cartilage differentiation seem to be exchanged during formation of ectopic cartilage. Muscle tissue and periosteum can be triggered to form cartilage by demineralized bone matrix. Chick limb bud epithelium induces type II collagen synthesis in embryonic mouse tooth germ, whereas homologous, oral epithelium induces the formation of dentin (type I collagen). Thus, the type of response elicited from mesenchyme cells can be determined by nearby epithelia, and that response frequently can be the formation of cartilage.