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R M Grainger

Publications and source records attributed to R M Grainger.

At least 55 records · Page 3Linked to original sources
PubMed1987

Inductive interactions in the spatial and temporal restriction of lens-forming potential in embryonic ectoderm of Xenopus laevis.

The process of lens cell determination in amphibians is currently viewed as one involving a series of inductive interactions. On the basis of previous investigations, these interactions are thought to begin during gastrulation when the presumptive foregut endoderm and then the heart mesoderm come into contact with the presumptive lens ectoderm. This earlier period of induction is followed by the later interaction of the optic vesicle with the lens-forming ectoderm. Transplantation experiments were performed to determine the relative significance of the early and later periods of induction in the process of lens cell determination in the anuran Xenopus laevis. Various ectodermal tissues were transplanted either into the lens-forming region of open neural plate stage host embryos or over the newly formed optic vesicle of later neurula stage embryos. All transplanted tissues were labeled with the intracellular marker horseradish peroxidase to assess the exact origins of any induced lens structures. The results indicate that all nonneural ectodermal tissues have some lens-forming potential early during gastrulation; however, this potential is restricted to the lens-forming region, and perhaps nearby regions, later in development during the time of neurulation. Furthermore, the results show that the optic vesicle is not a substantial inductor of the lens in tissues that have not been previously exposed to the earlier series of inductive interactions that take place during gastrulation and neurulation. Since the optic vesicle does not appear to be a sufficient inductor of the lens, these earlier inductive interactions are, therefore, essential in the process of lens cell determination in Xenopus. These earlier inductive interactions lead to a steady increase in what may be called a lens-forming bias in the presumptive lens ectoderm during this period of development. The eventual loss in the ability of nonlens ventral ectoderm to respond to these lens inductors is presumably the result of other determinative processes that occur in this tissue.

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PubMed1987

Delta-crystallin genes become hypomethylated in postmitotic lens cells during chicken development.

Although it has been argued that the loss of 5-methylcytosine from specific sites in DNA plays an important role in activation of specific genes, the mechanism of hypomethylation is not well understood. One model links the process to DNA replication, proposing that it occurs by not remethylating cytosine on newly synthesized DNA. An alternative model argues that hypomethylation results from excision of part or all of the 5-methylcytosine. We were able to test whether hypomethylation can occur without replication by analysis of methylation changes in the delta-crystallin genes of the chicken lens. During embryonic development a large fraction of cells in the lens stops dividing as part of the differentiation process. Shortly after this stage, the delta-crystallin genes in samples of the whole lens become hypomethylated, suggesting the possibility that this process might be occurring in the subset of cells that is no longer dividing. We found that hypomethylation of these genes does occur in postmitotic lens cells, a result that implicates an excision mechanism in this tissue.

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PubMed1986

Different protein synthetic patterns in scale-forming, feather-forming, and apteric embryonic chick dermis.

We have examined the protein synthetic profile of embryonic chick dermis from different regions of both wild-type and scaleless mutant embryos by two-dimensional polyacrylamide gel electrophoresis to determine if differences in inductive capability are associated with different patterns of gene expression. We have found proteins preferentially synthesized in dorsal dermis and anterior tarsometatarsal dermis at stages when these tissues are active in inducing feather or scale histogenesis, respectively, in the epidermis. Apteric dermis, which is unable to induce epidermal derivative formation, synthesizes a subset of the proteins specific to each region. Scaleless mutant dermis, which does not participate in feather or scale formation in vivo, synthesizes all of the dorsal dermis-specific or tarsometatarsal dermis-specific proteins appropriate to its regional origin. However, it lacks one protein common to all types of dermis tested, and synthesizes one protein inappropriate for its location. Examination of the protein synthetic profile of dorsal and anterior tarsometatarsal dermis at early stages of development reveals that young dorsal dermis, which can only form feathers, possesses the protein synthetic pattern specific to that region. Young tarsometatarsal dermis, which has the potential to form either feathers or scales, synthesizes the proteins we have identified as specific to dorsal and older tarsometatarsal dermis. These results suggest that different protein synthetic patterns are associated with different inductive potentials. However, combining young tarsometatarsal dermis with dorsal epidermis, which causes the formation of feathers, does not alter the pattern of proteins synthesized by the dermis. While this result may be due to an artifact of the culture system, an alternative explanation is that the protein synthesis pattern is not related to the type of epidermal derivative induced, but to the pattern in which the derivatives are induced. This is supported by the observation that the feathers formed in recombinants of tarsometatarsal dermis and dorsal epidermis are arranged in a scale pattern.

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PubMed1985

Drosophila ribosomal RNA stability increases during slow growth conditions.

We have developed density labeling pulse-chase methods which, in contrast to a conventional radiolabeling approach, allow us to determine the effectiveness of our chase and to measure RNA stability in vivo without measuring precursor pool specific activities. We have used these methods to determine the stability of the embryonic ribosomal RNA inherited by either normally or slowly growing Drosophila melanogaster larvae. If larvae are raised in a rich growth medium, embryonic rRNA decays with a half-life of 48 h. However, if larvae are raised in a poor growth medium, which slows larval growth and prolongs development, the half-life of rRNA increases to 115 h. This is the only example, of which we are aware, directly showing that rRNA half-life increases during slow growth conditions. We propose that the increased stability of rRNA that we find may enable slowly growing larvae to maintain the ribosome levels necessary to continue growth and development under conditions of nutrient deprivation.

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PubMed1985

Polyadenylation state of abundant mRNAs during Drosophila development.

We have used a two-dimensional gel analysis of cell-free translation products to determine whether individual mRNAs present in Drosophila melanogaster embryos, larvae, pupae, and adults are predominantly polyadenylated or nonadenylated. While the majority of the embryonic mRNAs we detected exist mainly in the polyadenylated form, these mRNAs become more evenly distributed between the poly(A)+ and poly(A)- RNA fractions during postembryonic development. Although DNA:RNA hybridization experiments have indicated that Drosophila RNA populations contain a large group of rare class mRNAs restricted to the poly(A)- RNA compartment, this is not true for the 150 more abundant mRNA species analyzed by our methods. The histone mRNAs are the only abundant mRNA species which appear to be exclusively in the poly(A)- RNA class.

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PubMed1985

Differentiation of embryonic chick feather-forming and scale-forming tissues in transfilter cultures.

The dermal-epidermal tissue interaction in the chick embryo, leading to the formation of feathers and scales, provides a good experimental system to study the transfer between tissues of signals which specify cell type. At certain times in development, the dermis controls whether the epidermis forms feathers or scales, each of which are characterized by the synthesis of specific beta-keratins. In our culture system, a dermal effect on epidermal differentiation can still be observed, even when the tissues are separated by a Nuclepore filter, although development is abnormal. Epidermal morphological and histological differentiation in transfilter cultures are distinct and recognizable, more closely resembling feather or scale development, depending on the regional origin of the dermis. Differentiation is more advanced when epidermis is cultured transfilter from scale dermis than from feather dermis, as assessed by morphology and histology, as well as the expression of the tissue-specific gene products, the beta-keratins. Two-dimensional polyacrylamide gel analysis of the beta-keratins reveals that scale dermis cultured transfilter from either presumptive scale or feather epidermis induces the production of 7 of the 9 scale-specific beta-keratins that we have identified. Feather dermis, although less effective in activating the feather gene program when cultured transfilter from either presumptive feather or scale epidermis, is able to turn on the synthesis of 3 to 6 of the 18 feather-specific beta-keratins that we have identified. However, scale epidermis in transfilter recombinants with feather dermis also continues to synthesize many of the scale-specific beta-keratins. Using transmission and scanning electron microscopy, we detect no cell contact between tissues separated by a 0.2-micron pore diameter Nuclepore filter, while 0.4-micron filters readily permit cell processes to traverse the filter. We find that epidermal differentiation is the same with either pore size filter. Furthermore, we do not detect a basement membrane in transfilter cultures, implying that neither direct cell contact between dermis and epidermis, nor a basement membrane between the tissues is required for the extent of epidermal differentiation that we observe.

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PubMed1985

Differential stability of Drosophila embryonic mRNAs during subsequent larval development.

The relative stabilities of specific embryonic mRNAs that persist in Drosophila melanogaster larvae were determined using an approach that combined RNA density labeling with cell-free translation. Unlike the other methods commonly used to measure the decay of individual mRNAs, the density labeling approach does not depend on the use of transcriptional inhibitors or on the measurement of precursor pool specific activities. Using this approach, we have determined that different embryonic mRNA species persist for varying periods during subsequent development, with half-lives ranging from approximately 2 to approximately 30 h. The embryonic histone mRNAs are relatively unstable; they are no longer detectable by 9 h of larval development. By 41 h of larval development, 90% of the nonhistone mRNAs assayed have decayed considerably; computerized scanning densitometry of translation products indicates that these transcripts are not decaying as members of discrete half-life classes. The persisting mRNAs that remain are very long-lived; their in vitro translation products can still be detected after 91 h of larval development. We have tentatively identified the mRNAs that encode actin, tropomyosin, and tubulin as members of this stable mRNA population. Although embryonic mRNAs do fall into these three broad classes of stability, they appear to decay with a continuum of half-lives. Because the range of half-lives is so great, mRNA stability is probably an important factor controlling mRNA abundance during Drosophila development.

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PubMed1981

A mosaicism in the higher order structure of Xenopus oocyte nucleolar chromatin prior to and during ribosomal gene transcription.

We report an analysis by electron microscopy of the differences in the folding of ribosomal gene and adjacent nontranscribed spacer DNA of Xenopus laevis oocytes into supranucleosomal chromatin structures. The chromatin structures identified in gene and spacer regions of transcriptionally active nucleoli (from stage 5 oocytes) were compared with those found in nucleoli prior to transcription (from stage 2 oocytes) to determine whether changes in the chromatin structure occur when transcription is initiated. Chromatin structures were characterized by their morphology and by the extent of folding of DNA in chromatin. Nontranscribed spacer regions from both transcriptionally active and inactive nucleoli appear to be packaged into supranucleosomal structures and are contracted by a factor of at least 20 from the length of B form DNA. The structure of the adjacent gene region, both before and during transcription, is much more extended; the only structures observed are the size of nucleosomes, and the DNA is contracted by a factor of 1.4 from its B form length. Thus a mosaicism in the higher order structure of gene and spacer rDNA is established days or weeks prior to the initiation of transcription of these genes and maintained during transcriptionally active stages.

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PubMed1980

A repeating unit of higher order chromatin structure in chick red blood cell nuclei.

The organization of nucleosomes in higher order chromatin structures has been studied by electron microscopy of chick red blood cell nuclei. Chromatin appears as a thick fiber with an average diameter of approximately 300 A when prepared for electron microscopy in buffers which approximate physiological ionic strength. Progressive steps of disassembly of the thick fiber into individual nucleosomes could be induced either by ionic strength reduction or by tRNA treatment (which removes histone H1 and some non-histone chromosomal proteins). When disassembly was induced by ionic strength reduction in the presence of Mg++ (or Ca++), the lengths of the intermediate disassembly products were found to be multiples of 330 A. The diameter of these structures was estimated to be 275 A. This intermediate in the disassembly process is not observed if thick fiber disassembly is induced by ionic strength reduction in the absence of divalent cations. To investigate whether the higher order structural unit is present in the thick fiber at physiological ionic strengths, tRNA treatment was used to induce thick fiber disassembly under physiological monovalent ionic conditions. In this case, either with or without divalent cations, a supranucleosomal unit was found with dimensions similar to those given above. This data provides evidence for a slightly oblong supranucleosomal structure (330 x 275 A) whick forms a repeating unit in the chromatin thick fiber.

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PubMed1980

Dictyostelium ribosomal RNA is processed during transcription.

Electron microscopy of transcriptionally active Dictyostelium ribosomal RNA genes reveals that ribosomal RNA processing occurs during transcription: each transcription unit is composed of two tandem ribonuclear protein matrices. This is the first example among eucaryotes of simultaneous transcription and processing of rRNA.

Dictyostelium↗
PubMed1979

Genetic modulation of RNA metabolism in Drosophila. IV. Measurement of rats of RNA synthesis by density labeling.

Accumulation of RNA was measured in adult males of two genotypes: car bb/Ybb- and car bb/YbbSuVar-5. The two genotypes have similar amounts of rDNA, which is reduced in comparison to wild type (CLARK, STRAUSBAUGH and KIEFER 1977). Although genotypically bobbed, car bb/YbbSuVar-5 flies have a wild-type phenotype; car bb/Ybb- flies are both phenotypically and genotypically bobbed (CLARK, STRAUSBAUGH and KIEFER 1977). The wild-type phenotype observed in the car bb/YbbSuVar-5 flies is thought to be the result of an increased rate of rRNA synthesis due to the presence of the YbbSuVar-5 chromosome (SHERMOEN and KIFFER 1975; CLARK, STRAUSBAUGH and KIEFER 1977; CLARK and KIEFER 1977). To further define this phenomenon, the absolute accumulation of RNA was measured in the two genotypes, using density labeling methods. The accumulation of RNA is 1.4 to 1.8 times higher in car bb/YbbSuVar-5 flies than in car bb/Ybb- flies, demonstrating that there is genetic regulation of synthesis in this genotype. The use of density-labeled nucleosides has clearly shown that there is no difference in precusor pool sizes or use between the two genotypes studied.

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