Sarah C.R. Elgin

The chromatin structure of specific genes: I. Evidence for higher order domains of defined DNA sequence.

Abstract When the chromatin of Drosophila is examined by digestion with DNAase I or micrococcal nuclease, no general structural organization above the level of the nucleosome is revealed by the cleavage pattern. In contrast, the DNAase I cleavage pattern of specific regions of the Drosophila chromosome shows discrete bands with sizes ranging from a few kilobase pairs (kb) to more than 20 kb. Visualization of such higher order bands was achieved by the use of the Southern blotting technique. The DNAase I-cleaved fragments were transferred onto a nitrocellulose sheet after size fractionation by gel electrophoresis. Hybridization was then carried out with radioactively labeled cloned fragments of DNA from D. melanogaster. For the five different chromosomal regions examined, each gives a unique pattern of higher order bands on the autoradiogram; the patterns are different for different regions. Restriction enzyme cleavage of the fragments generated indicates that the preferential DNAase I cleavage sites in chromatin are position-specific. The chromosomal regions bounded by preferential DNAase I cleavage sites are referred to as supranucleosomal or higher order domains for purposes of discussion and analysis. The micrococcal nuclease cleavage pattern of chromatin at specific loci was also examined. In the one case studied in detail, this nuclease also cleaves at position-specific sites.

The chromatin structure of specific genes: II. Disruption of chromatin structure during gene activity

We have compared the chromatin structure in the active and inactive states at loci encoding the major heat shock protein in Drosophila. DNAase I and micrococcal nuclease were used as probes of higher order organization and nucleosomal integrity. Such integrity is gauged here by the characteristic pattern of discrete DNA fragments produced at specific chromosomal loci by nucleolytic cleavage. The specific fragment patterns are visualized by gel electrophoresis, Southern blotting onto nitrocellulose sheets, hybridization with 32P-labeled cloned DNA containing the heat shock genes and autoradiography. Using this criterion, a disruption in nucleosomal and possibly in higher order organization are observed as indicated by a relative loss or smearing of the characteristic discrete DNA fragment patterns from the heat shock loci in the active state. The fragment patterns are restored when cells are allowed to recover from heat shock and these loci return to the inactive state.

A protein released by DNAase I digestion of drosophila nuclei is preferentially associated with puffs

Antisera have been produced against five molecular weight subfractions of the Drosophila proteins readily extracted from nuclei following limited DNAase I digestion. Immunofluorescence staining techniques were used to assess the distributions of these proteins in the polytene chromosomes of Drosophila. In three cases, the antigens were widely distributed; in one case, the antigens appeared to be slightly more concentrated at active loci; and in one case, the antigens were strongly concentrated at a defined set of loci, including puffs and most of the loci which are active (puffed) at some time during third instar larval and prepupal development. The latter distribution pattern differs from that of RNA polymerase. Nonhistone chromosomal proteins of this type may have a key role in establishing and/or maintaining the altered chromatin structure characteristic of the active state.

Distribution patterns of three subfractions of drosophila nonhistone chromosomal proteins: possible correlations with gene activity

Abstract The distribution of three molecular weight subfractions of the Drosophila nonhistone chromosomal proteins (NHC proteins) has been studied using an immunofluorescent technique (Silver and Elgin, 1976). In all three cases, the fluorescence distribution patterns obtained are distinct and reproducible. The results imply that different NHC protein components have different distributions along the polytene chromosomes. A highly selective pattern is obtained using antiserum against subfraction ϱ; puffs (loci highly active in RNA synthesis) and many nonpuffed chromomeres which are known to puff at other times during the third larval instar or prepupal stages are brightly fluorescent. New RNA synthesis can be induced at 87A, 87B-C1 and other chromomeres by heat shock treatment; these loci, previously stained at low levels, are subsequently stained brightly using the ϱ serum. The staining of the heat shock puffs appears to be superimposed upon the prior ϱ pattern. The results suggest that a change in chromosomal structure, as indicated by staining using the ϱ serum, is associated with gene activity as indicated by puffing. This different chromosomal structure may be the consequence of either a redistribution of a ϱ antigenic determinant [a new association of specific protein(s) with the active sites] or a change in chromatin configuration [making the ϱ antigenic determinant(s) newly available to the antibody probe].

The proteins of polytene chromosomes of Drosophila hydei

It is reported that chromatin can be prepared from highly purified polytene nuclei from the salivary glands of third instar larvae of Drosophila hydei; such chromatin differs from that of diploid nuclei mainly by deficiencies in certain nonhistone chromosomal proteins. It is suggested that these proteins are important components of constitutive heterochromatin, which is severely underrepresented in polytene chromosomes. Chromosome morphology, including the pattern of induced puffs, is maintained throughout the mass isolation of glands and sucrose gradient purification of nuclei, as indicated by studies on temperature-shocked and control larvae. No significant alteration in the chromosomal proteins following puff induction by heat shock could be detected on analysis of the isolated protein fractions by disc gel electrophoresis. More sensitive techniques must be developed to study the apparent rearrangement or accumulation of protein at puff sites, and to elucidate the role of this protein in gene activation.

Chapter 10 Immunofluorescent Techniques in the Analysis of Chromosomal Proteins

Publisher Summary This chapter focuses on immunofluorescent techniques in the analysis of chromosomal proteins. Progress in the analysis of the nonhistone chromosomal proteins (NHC proteins) has been slow because of the complexity of this class of proteins and the fact that many of the major NHC proteins apparently play structural roles for which no direct assay is available. The use of specific antibody techniques appears likely to assist in resolving these problems. Specific antibodies against chromosomal proteins can be used to determine the distribution of these proteins in situ using indirect staining techniques. The in situ distribution patterns obtained can suggest as well as test hypotheses concerning structural and active roles of these proteins. Specific antibodies can also be used for rapid purification of components from complex mixtures and as probes in enzyme assays. The chapter discusses the production of antibodies against chromosomal proteins of Drosophila and their use in an indirect immunofluorescent assay to determine the in situ distribution of these proteins. The chapter discusses the preparation of antigens and antisera and the characterization of antiserum by indirect immunofluorescent staining of sodium dodecyl sulfate (SDS)-polyacrylamide gels.

Production and characterization of antisera against three individual NHC proteins; a case of a generally distributed NHC protein

In order to assess the selectivity of the distribution patterns of individual nonhistone chromosomal proteins (NHC proteins), immunofluorescent staining experiments were performed on Drosophila polytene chromosomes. Antisera have been prepared against three individual NHC proteins which were isolated by sequential preparative slab gel isoelectric focusing and SDS polyacrylamide gel electrophoresis. In two cases, immunofluorescent staining of the chromosomes indicated a specific limited distribution pattern; apparently the antigen in each case is present at a reproducible and distinct subset of chromomeres. This type of pattern has also been obtained with antisera prepared against molecular weight subfractions of NHC proteins (Silver and Elgin, 1977). Each selective fluorescence distribution pattern obtained so far is reproducible and unique to the antiserum under study. In a third case, an antiserum caused prominant staining at dense chromomeres and the chromocenter in a pattern mimicking DNA (and presumably histone) distribution. Indirect radioimmunostaining of SDS and isoelectric focusing gels on which total NHC proteins had been separated confirmed that this antiserum reacted specifically with a protein(s) of molecular weight 21,000 D and pI 5.2. The data in conjunction with absorption experiments indicates that the chromosomal staining is due to an interaction of antibodies with NHC protein(s) and not with histones. This finding suggests that at least one major acidic NHC protein plays a very general role (comparable to that of the histones) in maintaining chromatin structure.

Chapter 4 Immunofluorescent Analysis of Chroma Structure in Relation to Gene Activity: A Speculative Essay

Publisher Summary Immunofluorescence procedures to analyze the in situ distribution patterns of specific chromosomal proteins on Drosophila polytene chromosomes have been developed in past years. This chapter focuses on a set of results that have been obtained with this technique indicating the distribution patterns of nonhistone chromosomal (NHC) proteins, which may reflect structural differences in chromatin related to the control of gene expression. The technique of in situ hybridization is used to localize the complements of particular DNA and RNA sequences to particular polytene chromosome bands. By using antisera against specific chromosomal proteins, the pattern of distribution of these components in relation to the established banding pattern can be similarly assessed. It is of particular interest to consider the distribution pattern of chromosomal proteins in relation to the patterns of gene activity. The evidence for at least three types of NHC proteins bearing different relationships to active and inactive genes is relatively convincing—namely, (1) those widely distributed in chromatin, (2) those associated with the developmentally active loci and heat shock-activated loci of the third instar salivary gland, and (3) those associated primarily with active loci such as RNA polymerase II.

An immunofluorescent analysis of Drosophila polytene chromosomes with antisera directed against H3, H4, and a native H3–H4 complex

Abstract Fixed polytene chromosome preparations have been stained by indirect immunofluorescence. Anti-H3 serum and anti-H4 serum cause very intense and highly specific staining of chromosomes. Anti-(H3–H4 complex) serum did not produce staining of chromosomes at a level above background. The results obtained in these staining experiments are in direct contrast to serological results obtained with soluble chromatin. It appears that a unique structure exists within the H3–H4 complex that is not present on the individual histone components. This structure is apparently destroyed or obscured by acetic acid fixation during the preparation of polytene chromosome spreads.

5'-Mercuri-uridine triphosphate as a substrate for transcription.

Abstract The use of mercurated nucleotides permitting a specific isolation of newly synthesized RNA has proven useful in the study of in vitro transcription. However, there has been little direct investigation of their effect on the polymerization reaction. We have synthesized 5′-mercuri-uridine [α- 32 P]triphosphate and analyzed its use in an in vitro nuclear transcription system. Nearest-neighbor analysis of the product was performed. Comparison of the frequencies obtained in this case to those obtained when uridine [α- 32 P]triphosphate or iodouridine [α- 32 P]triphosphate was used indicates that premature termination is occurring at a mercuri-uridine residue; the data suggest that this result is due predominantly to the size of the analog rather than to a specific chemical effect. Such premature termination occurs most frequently when a mercuri-uridine residue follows a guanosine residue.