Differential regulation of homologous chromosomes in a mealybug : planococcus citri chromosomanal proteins and DNA

Abstract

We have been interested in the regulatory events that initiate and maintain the inactivation of an entire set of chromosomes in the males of the mealybug Planococcus citri. This genetic system is nearly unique because an entire haploid genome (paternal) is inactive, whereas the homologous maternal genome within the same nucleus is transcriptionally active or potentially active. No such inactivation occurs in females. This system has analogies in Sciara, mammals, cecidomyiidae, and possibly also in Ascaris. The mechanism controlling this differential regulation of homologous chromosomes is of considerable current interest (see, for example, Sager, R. and Ritchin, R. (1975) Science 189, 426–453; Riggs, A.D. (1975) Cytogenet. Cell Genet. 14, 9–25) and is being viewed as a developmental problem with wide implications. As a beginning to our understanding of this problem, we have characterized the chromosomal proteins of this insect. Although there is no significant difference between the core histones of males and females, the pattern of H1 histones was different. Two different degrees of H1 phosphorylation were observed:

A basal level of phosphorylation present in both males and females, and A hyperphosphorylated form of H1, present only in males.

The varying degrees of H1 phosphorylation were detected by alkaline phosphatase time dependent digestion. The exclusive presence of hyperphosphorylated H1 in males indicates that this form may be involved in the compaction of the inactive paternal set into facultative heterochromatin. To determine whether this difference persists at the level of fractionated chromatin, we standardized conditions for obtaining maximal separation of mealybug chromatin into:

a fraction enriched in transcriptionally active chromatin, and a fraction enriched in transcriptionally inactive chromatin.

These two fractions have been characterized on the basis of their spectral properties, melting profiles, and ability to act as templates for E. coli DNA dependent RNA polymerase. Other properties of these fractions, such as DNase I sensitivity and the proteins associated with them, have also been examined. The High Mobility Group (HMG) of proteins from these insects has been characterized, and differences in their distribution between the active and inactive fractions have been examined. The hyperphosphorylated H1 was found to be selectively associated with inactive chromatin. This supports our assumption that this form of H1 is very likely involved in the condensation of facultative heterochromatin. In mealybugs, Sciara, and marsupials, it is the paternal chromosomes that are selectively inactivated or eliminated. How do chromosomes know which parent they come from The term chromosome imprinting was introduced (Crouse, 1960, Genetics 45, 1429–1443; Brown and Chandra, 1975, Nature 254, 165–168) to denote the molecular event, or set of events, that determines that a chromosome will behave differently from its homolog in the same nucleus at a subsequent stage in development. The nature of this molecular change is not known, but several interesting models have been proposed, of which the methylation model is perhaps the most favored. Methylation is known to affect various physical properties of DNA such as buoyant density. We therefore subjected both male and female DNAs to CsCl equilibrium density centrifugation. Over 90% of the female DNA banded as a single symmetric peak at a density of 1.691 g/cm³. DNA from males, on the other hand, banded as two symmetric peaks. The lighter peak had a density identical to that of female DNA. The remainder of the male DNA, about 50%, banded as an altogether new peak at 1.705 g/cm³. Melting studies failed to show any difference in the G+C content of male and female DNAs, suggesting that the separation of male DNA into two species is not due to differences in GC content. The bases were analyzed by TLC; two additional bases were detected and were identified as 5 methylcytosine and 6 methyladenine, based on their spectral characteristics. The proportion of modified bases in each sex was quantified by HPLC. We estimate an increase of about 10% (on a molar basis) of methylated bases in female DNA compared to that of males. The difference in methylation levels between male and female DNAs as studied by HPLC is in general agreement with the buoyant density data. A model based on chromosome imprinting by methylation and subsequent condensation and inactivation of the paternal set by H1 phosphorylation has been proposed. Attempts have been made to establish primary cell cultures from mealybug tissues. After many unsuccessful attempts, we have been able to obtain reasonably good growth of these cells in a 1:1 mixture of TC 199 and Mitsuhashi–Maramorosch medium (MM), pH 7.0, at 28°C. However, the population doubling time of these cells is 10–15 days, compared to that of other insect cells, which is about 72 hours. Attempts are now being made to improve and enhance the growth of these cells in culture. It is obvious that a reliable method for culturing these cells would permit many rigorous experiments to be undertaken to understand the molecular basis of this unusual genetic system.