Charles D. Laird

Chromatid structure: relationship between DNA content and nucleotide sequence diversity.

Models of chromatid structure are based on inferences made from genetic, cytological, and cytochemical observations. An alternative approach can provide limits as to the number of identical subunits present in chromatids. This method is based on the demonstration that nucleotide sequence diversity may be estimated from the kinetics of renaturation of denatured DNA. Measurements of DNA content and renaturation rate constants are given for several eukaryotic DNAs. Control experiments involved measurements of renaturation kinetics of DNAs from bacteria and bacteriophage. These estimates show that most of the nucleotide sequences in mouse, Drosophila, and Ciona DNA are present only once per sperm. Since the reduction of DNA content during meiosis indicates that mouse sperm contain a haploid set of chromatids, it follows that a set of mouse meiotic chromatids contains a single copy of most sequences. Models of chromatid structure which postulate multiple subunits with identical nucleotide sequences are therefore not tenable for mouse meiotic chromatids. This method of analyzing nucleotide sequence diversity may be of general use in designing models of chromatid structure in other organisms.

Radiation-sensitivity of bacteriophage containing 5-bromodeoxyuridine☆☆☆

Abstract Bacteriophage containing 5-bromodeoxyuridine have increased sensitivity to X-rays, ultraviolet light (2537 A), and light of longer wavelengths emanating from commercial fluorescent fixtures. The increase in sensitivity for the last case amounts to a factor of 75 for phage T2 u when fully substituted. A proffered hypothesis to account for the increased sensitivity is based on a comparative study of UV-resistant and UV-sensitive strains of T2.

Hairpin-bisulfite PCR: Assessing epigenetic methylation patterns on complementary strands of individual DNA molecules

Epigenetic inheritance, the transmission of gene expression states from parent to daughter cells, often involves methylation of DNA. In eukaryotes, cytosine methylation is a frequent component of epigenetic mechanisms. Failure to transmit faithfully a methylated or an unmethylated state of cytosine can lead to altered phenotypes in plants and animals. A central unresolved question in epigenetics concerns the mechanisms by which a locus maintains, or changes, its state of cytosine methylation. We developed “hairpin-bisulfite PCR” to analyze these mechanisms. This method reveals the extent of methylation symmetry between the complementary strands of individual DNA molecules. Using hairpin-bisulfite PCR, we determined the fidelity of methylation transmission in the CpG island of the FMR1 gene in human lymphocytes. For the hypermethylated CpG island of this gene, characteristic of inactive-X alleles, we estimate a maintenance methylation efficiency of ≈0.96 per site per cell division. For de novo methylation efficiency (Ed), remarkably different estimates were obtained for the hypermethylated CpG island (Ed = 0.17), compared with the hypomethylated island on the active-X chromosome (Ed < 0.01). These results clarify the mechanisms by which the alternative hypomethylated and hypermethylated states of CpG islands are stably maintained through many cell divisions. We also analyzed a region of human L1 transposable elements. These L1 data provide accurate methylation patterns for the complementary strand of each repeat sequence analyzed. Hairpin-bisulfite PCR will be a powerful tool in studying other processes for which genetic or epigenetic information differs on the two complementary strands of DNA.

Comparative organization of active transcription units in Oncopeltus fasciatus

We have analyzed electron micrographs of chromatin-associated fiber arrays from embryos of the milkweed bug, Oncopeltus fasciatus. The analysis has revealed that the arrays have highly ordered patterns of fiber spacings and lengths. These patterns support the interpretation that the fibers are nascent RNA with associated proteins (RNP fibers) which have resulted from transcription of the DNA in the underlying chromatin segment. In particular, the patterns indicate that the chromatin underlying each array is delimited by specific sites for initiation and termination of transcription. We apply the term transcription unit to a chromatin segment thus bounded. The analysis has further revealed that transcription units can be grouped into two principal classes--ribosomal and nonribosomal. Active transcription units of these two classes differ in DNA content, in their proximity to other active transcription units, and in their chromatin morphology. For certain developmental stages, fiber frequencies (that is, the nubmers of fibers per mum of chromatin) are also useful in distinguishing ribosomal from nonribosomal arrays. The most definitive of the above classification criteria is chromatin morphology as observed under our preparative conditions. We propose that term rho chromatin for the unbeaded or smooth chromatin that underlies nascent ribosomal RNP fibers. DNA in rho chromatin has a calculated packing ratio of approximately 1.2 mum of B structure DNA per mum of chromatin. Nu chromatin is used to designate the beaded chromatin for which we calculate a DNA packing ratio of 1.6-2.3 in our preparations. This calculation for nu chromatin is based on the inference that the beads are nucleosomes (nu bodies, PS particles, unit particles). The beaded morphology is observed between fibers of nonribosomal transcription unit as well as for most fiber-free chromatin. The detection of specific sites of transcriptional initiation and termination and the classification of transcription units can provide a basis for further analysis of transcriptional control.

Fragile sites in human chromosomes as regions of late-replicating DNA

Abstract We review data indicating that fragile sites in chromosomes of humans, Drosophila and Microtus represent regions where DNA is late-replicating in the cell cycle. We suggest that rare fragile sites in human chromosomes represent cis -acting alterations to DNA that confer or accentuate late replication at that site. The possible connection between late replication and the fragile X syndrome is also discussed.

Transcription of Nonrepeated DNA in Mouse Brain

Under normal conditions of DNA renaturation, about 60 percent of mouse DNA fragments renature at a rate consistent with their being present only once per sperm. These nonrepeated sequences (also called single-copy or unique) may be used in RNA-DNA hybridization experiments to provide quantitative estimates of RNA diversity. About 10 percent of the mouse single-copy sequences are transcribed in mouse brain tissue. Estimates of about 3 percent were obtained for mouse liver and kidney RNAs. If only one of the complementary DNA strands is transcribed, this hybridization value implies that the equivalent of at least 300,000 different sequences of 1000 nucleotides are expressed in mouse brain tissue. It is suggested that the large amount of DNA in mammals is functionally important, and that a substantial proportion of the genome is expressed in the brain.

Sequence diversity of polytene chromosome DNA from Drosophila hydei

Abstract Polytene chromosomes in salivary glands of Drosophila hydei contain as much as 660 times the amount of DNA present in non-polytene chromosomes (Mulder, van Duijn & Gloor, 1968). Information on the structure of these giant chromosomes is required for an understanding of the relationship between cytological and genetic units. We have used renaturation kinetics of DNA fragments (500 nucleotides long) to compare nucleotide sequence diversities in DNAs from polytene and non-polytene cells of D. hydei . These experiments show that DNAs from D. hydei pupae and from embryonic nuclei renature as if 80% of the nucleotide sequences are present only once per haploid cell. These sequences are therefore termed single-copy (also unique, or non-repeated) under the criteria imposed by our reaction conditions. To relate sequence diversity of DNA from polytene chromosomes to that inferred for haploid cells, DNA was isolated from salivary gland nuclei. DNA fragments from this source renature with kinetics similar to those observed for single-copy DNA from pupae and embryos. We conclude that most of the single-copy sequences present in non-polytene chromosomes of D. hydei undergo essentially equal replication in the formation of salivary gland polytene chromosomes. This extent of sequence diversity does not support models of gene organization that depend on a general and high degree of intrachromatid redundancy. Polytene chromosome DNA does differ from non-polytene chromosome DNA, however, in that it contains about 5% fast-renaturing sequences, instead of the 20% present in embryo and pupal DNAs. Cytogenetic observations and cytochemical measurements imply that DNA in centromeric heterochromatin is under-replicated during formation of Drosophila polytene chromosomes (Painter, 1934; Heitz, 1934; Mulder et al. , 1968; Rudkin, 1969; Gall, Cohen & Polan, 1971). Since the fast-renaturing components are also under-represented in polytene DNA, we suggest that most of the repeated sequences in D. hydei are localized near the centromeres. Conversely, the retention of sequence diversity of slowly renaturing fragments implies that these single-copy sequences are predominantly localized in euchromatin. These data place limits on models of DNA organization in chromosomes of Drosophila .

Increase in nuclear poly(A)-containing RNA at syncytial blastoderm in Drosophila melanogaster embryos.

Abstract Levels of poly(A)-containing RNA in embryonic nuclei of Drosophila melanogaster were related to the morphological events of nuclear migration and cell formation. Poly(A)-containing RNA was detected by in situ hybridization of [ 3 H]poly(U) to cytological preparations of ruptured embryos. Increased levels of [ 3 H]poly(U) binding were observed in nuclei that had migrated to the cortical cytoplasm. Ribonuclease-treated preparations indicated that increased poly(U) binding was RNA dependent. Pole cells, which are the first cells formed, did not show this increase in binding. We interpret these data as evidence that transcriptional activation of informational sequences occurs shortly after nuclei have migrated to the cortical cytoplasm and before cell membranes are completed. The absence of this activation in nuclei of pole cells may result from repressor activity in the polar plasm.

Chromosome structure and DNA replication in nurse and follicle cells of Drosophila melanogaster

In the nurse cells of Drosophila, nuclear DNA is replicated many times without nuclear division. Nurse cells differ from salivary gland cells, another type of endoreplicated Drosophila cell, in that banded polytene chromosomes are not seen in large nurse cells. Cytophotometry of Feulgen stained nurse cell nuclei that have also been labeled with 3H-thymidine shows that the DNA contents between S-phases are not doublings of the diploid value. In situ hybridization of cloned probes for 28S+18S ribosomal RNA, 5S RNA, and histone genes, and for satellite, copia, and telomere sequences shows that satellite and histone sequences replicate only partially during nurse cell growth, while 5S sequences fully replicate. However, during the last nurse cell endoreplication cycle, all sequences including the previously under-replicated satellite sequences replicate fully. In situ hybridization experiments also demonstrate that the loci for the multiple copies of histone and 5S RNA genes are clustered into a small number of sites. In contrast, 28S+18S rRNA genes are dispersed. We discuss the implications of the observed distribution of sequences within nurse cell nuclei for interphase nuclear organization. — In the ovarian follicle cells, which undergo only two or three endoreplication cycles, satellite, histone and ribosomal DNA sequences are also found by in situ hybridization to be underrepresented; satellite sequences may not replicate beyond their level in 2C cells. Hence the pathways of endoreplication in three cell types, salivary gland, nurse, and follicle cells, share basic features of DNA replication, and differ primarily in the extent of association of the duplicated chromatids.

Morphology of transcription units inDrosophila melanogaster

We have used an electron microscopic analysis to define and to characterize active transcription units ofDrosophila melanogaster. The lengths and spacings of nascent ribonuclear protein (RNP) fibers were determined on embryonic chromatin that was spread using techniques introduced by Miller and Beatty (1969). The data are consistent with the occurrence of specific sites of transcription initiation and termination. We apply the termtranscription unit (TU) to a chromatin region bounded by these control sites. Two classes of TUs are active inDrosophila melanogaster embryonic cells—those synthesizing ribosomal RNA and those synthesizing non-ribosomal RNA. The classes can usually be distinguished on the basis of TU size, chromatin morphology and inferred DNA packing ratio, frequency of RNP fibers (number of fibers per μm of chromatin), and the solitary vs. tandem repeat occurrence of fiber arrays. The results indicate that nonribosomal transcription units have lengths in accord with the expectation that DNA of each chromomere is transcribed as a unit.—Some nascent fiber arrays inD. melanogaster have more complex patterns of RNP fiber lengths. We suggest that these are a consequence of cleavage of RNP fibers at specific sites during transcription. — These sites of transcriptional control and the amounts of DNA between them provide a basis for further relating units of transcription to units of gene function.