Marx K A, Assil I Q, Bizzaro J W, Blake R D
Department of Chemistry, University of Massachusetts, Lowell 01854, USA.
J Biomol Struct Dyn. 1998 Oct;16(2):329-39. doi: 10.1080/07391102.1998.10508250.
The slime mold, Dictyostelium discoideum, possesses an (A+T) rich eukaryotic genome that is being sequenced in the Human Genome Project. High resolution melting curves of isolated total and fractionated nuclear D. discoideum DNA(AX3 strain) were determined experimentally and are compared to melting curves calculated from GENBANK sequences (1.59% of genome) by the statistical thermodynamics program MELTSIM (1), parameterized for long DNA sequences (2,3). The lower and upper temperature limits of calculated melting agree well with the observed melting of total DNA. The experimental curve is unusual in that it contains a number of sharp peaks. MELTSIM allowed us to calculate positional denaturation maps of D. discoideum GENBANK sequence documents containing the 26S, 5.8S and 17S rDNA gene sequences, a major satellite DNA and repetitive sequence family present in 100-200 copies/nucleus. These denaturation maps contain subtransitions that correspond with a number of the experimentally observed peaks, some of which we show to correspond with rDNA gene enriched CsCl gradient fractions of D. discoideum DNA. MELTSIM calculated curves of coding, intron and flanking sequences indicate that both intron and flanking sequences are extremely (A+T) rich and account for most of the low temperature melting. There is no temperature overlap between thermal stabilities of these sequence domains and those of coding DNA. The latter must satisfy triplet codon constraints of higher (G+C) content. These large stability property differences enable a denaturation mapping feature of MELTSIM to clearly distinguish exon positions from those of introns and flanking DNA in long D. discoideum gene containing sequences.
黏菌盘基网柄菌拥有一个富含（A+T）的真核基因组，该基因组正在人类基因组计划中进行测序。通过实验测定了分离出的盘基网柄菌（AX3菌株）总核DNA和分级核DNA的高分辨率熔解曲线，并将其与通过统计热力学程序MELTSIM（1）根据GENBANK序列（占基因组的1.59%）计算出的熔解曲线进行比较，该程序针对长DNA序列进行了参数化（2,3）。计算出的熔解下限和上限温度与观察到的总DNA熔解情况吻合良好。实验曲线不同寻常之处在于它包含许多尖锐的峰。MELTSIM使我们能够计算盘基网柄菌GENBANK序列文档的位置变性图谱，这些文档包含26S、5.8S和17S rDNA基因序列、一种主要的卫星DNA和以100 - 200个拷贝/细胞核存在的重复序列家族。这些变性图谱包含与一些实验观察到的峰相对应的亚转变，我们发现其中一些峰与盘基网柄菌DNA的富含rDNA基因的CsCl梯度级分相对应。MELTSIM计算出的编码序列、内含子序列和侧翼序列的曲线表明，内含子序列和侧翼序列都极度富含（A+T），并且占低温熔解的大部分。这些序列结构域的热稳定性与编码DNA的热稳定性之间没有温度重叠。后者必须满足更高（G+C）含量的三联体密码子限制。这些大的稳定性差异使得MELTSIM的变性图谱特征能够在包含长盘基网柄菌基因的序列中清楚地将外显子位置与内含子和侧翼DNA的位置区分开来。
