Immunofluorescent techniques in the analysis of chromosomal proteins.
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Biomedical subjects
Publications and source records attributed to S C Elgin.
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A technique has been developed for "staining" cytological preparations by indirect immunofluorescent methods that permits determination of the in situ distribution of chromosomal proteins. The method is particularly oriented to the use of polytene chromosome squashes from Drosophila salivary glands. Control experiments indicate that the fixation methods used allow little or no extraction or rearrangement of the chromosomal proteins. The results obtained demonstrate the specific in vivo chromosomal locations of nonhistone proteins purified from isolated chromatin. The technique is apparently capable of resolution at the level of the chromomere or band, the unit of genetic organization in Drosophila.
The nonhistone chromosomal proteins (NHC proteins) probably include enzymes of chromosomal metabolism, general structural proteins, and possibly control elements. In theory, these proteins may have been strongly conserved during evolution, as the histones have. We have used sodium dodecyl sulfate (SDS) disc gel electrophoresis to analyze and compare the NHC proteins of two tissues, liver and kidney, from rat, cat, cow, chicken, turtle, and frog. The gel patterns indicate that the NHC proteins have changed much more during evolution than have the histones; the total pattern of NHC proteins has not been conserved. However, there does appear to be a conservation of a subset of bands for each tissue investigated. Further chemical analysis will be required to establish the significance of the results.
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.
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The multigene family is a unit of chromosomal organization. Its gene members are closely linked, homologous in sequence, and have overlapping functions. Multigene families can be divided into three catagories: simple-sequence, multiplicational, and informational-by a variety of structural and functional criteria. Multigene families exhibit two novel evolutionary features-coincidental evolution and rapid change in family size-that suggest that they all share one or more evolutionary mechanisms. Natural selection cannot act directly upon individual genes in a family because of their identical or overlapping functions; hence selection must operate upon the family as a whole or upon blocks of genes within the family. The mechanism(s) for coincidental evolution expands out variant genes within a family so they can be acted upon by natural selection and, accordingly, permits multigene families to evolve adaptively. The control mechanisms in multiplicational families appear to promote the rapid expression of many gene copies. In contrast, the regulatory mechanisms of informational families promote the selection, expression, and amplification of appropriate units of information. The close linkage of the genes in a family appears to be a consequence of the fact that their control and evolutionary mechanisms may only operate on tandemly linked genes. New multigene families may evolve from a single gene or from other multigene families. In addition to evolving new functions, the latter mode of evolution generates a new multigene family whose members are preadapted to interact with those of the old family. These family interactions can lead to the evolution of more sophisticated molecular machines or to the regulation of one family by a second. Multigene families may be large or small. The three catagories of multigene families allow potential multigene families to be identified, and they suggest specific experimental approaches for the study of new families. Some of the most interesting genetic systems under the investigation today are known or potential informational multigene families. This is not fortuitous in that many of the most interesting aspects of phenotype are complex ones with correspondingly complex genetic, evolutionary, and regulatory requirements. One of the frontiers in modern genetics is the identification, characterization, and understanding of informational multigene families.
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