H. R. Whiteley

Differential expression of the msp130 gene among skeletal lineage cells in the sea urchin embryo: a three dimensional in situ hybridization analysis

In order to examine the ontogeny of tissue-specific expression of the msp130 gene during early embryogenesis of the sea urchin, we have developed a whole-mount, non-radioactive in situ hybridization protocol suitable for these embryos. This protocol is adapted from the existing technology of immunohistochemical localization of digoxygenin-labelled hybridization probes in tissue sections. Transcript distribution patterns in the whole embryo are seen in three dimensions, and at much higher resolution and sensitivity than can be achieved using radioactive probes and sectioned material. We have traced the ontogeny of expression of the skeleton-specific gene, msp130, during the development of Strongylocentrotus purpuratus. Transcripts are first detected at the blastula stage, in micromere-lineage cells just prior to ingression. Appearance of msp130 transcripts remains strictly limited to this lineage through the pluteus stage. Estimated from the relative intensity of staining of the PMCs of an embryo, the relative abundance of msp130 transcripts is uniform among the 32 cells of this lineage in secondary mesenchyme blastulae and in gastrulae, indicating that expression is homogeneous among these cells up to the early prism stage. However, the relative intensity of stain, and therefore abundance of transcripts, changes dramatically and in a consistent pattern among the PMCs of an embryo during prism and pluteus stages, suggesting that these cells switch from an autonomous mode of regulation of the msp130 gene, to an inductive mode. In the pluteus larva, the highest levels of expression occur in those cells associated with the rapidly growing tips of the spicular skeleton.

Ferredoxin-dependent reactions in Micrococcus lactilyticus.

Abstract Evidence had been presented earlier (Whiteley and Ordal, 1956) that an iron-containing protein functions as an electron carrier in the dehydrogenation of hypoxanthine to xanthine and hydrogen by extracts of Micrococcus lactilyticus . An iron-containing protein, named ferredoxin, has recently been isolated and purified from Clostridium pasteurianum (Mortenson et al. , 1962) and M. lactilyticus (Valentine et al. , 1962) and has been found to participate in several hydrogenase-coupled reactions. The evolution of hydrogen from hypoxanthine by M. lactilyticus , the hydrogenase-linked reduction of nitrite and hydroxylamine by C. pasteurianum , and the production of hydrogen from pyruvate and dithionite by both anaerobes have been shown to require ferredoxin (Mortenson et al. , 1962 ; Valentine et al. , 1962) . Ferredoxin also mediates the photoevolution of hydrogen by Chromatium and the photosynthetic reduction of TPN by chloroplasts (Tagawa and Arnon, 1962) ; ferredoxin isolated from the latter source differs slightly in properties from the bacterial ferredoxin. Extracts of M. lactilyticus catalyze the evolution of hydrogen from several organic compounds and are able to utilize hydrogen to reduce a variety of dyes, inorganic anions, organic acids, purines and coenzymes. These reactions have now been examined with respect to ferredoxin dependency.

Coordinate accumulation of five transcripts in the primary mesenchyme during skeletogenesis in the sea urchin embryo

The sea urchin larval skeleton is produced by the primary mesenchyme (PM), a group of 32 cells descended from the four micromeres of the 16-cell embryo. The development of this lineage proceeds normally in isolated cultures of micromeres. A complementary DNA (cDNA) library was generated from cytoplasmic polyadenylated RNA isolated from differentiated micromere cultures of Strongylocentrotus purpuratus. Five clones were selected on the basis of their enrichment in differentiated PM cell RNA as compared to the polyribosomal RNAs of other embryonic cell types and other developmental stages. Each cloned cDNA hybridized to a distinct RNA that was abundant in the polyribosomes of differentiated PM cells, but absent from larval ectoderm and from 16-cell embryos. These RNAs were encoded by single or low copy genes. In situ hybridization analysis of the most abundant of these RNAs (SpLM 18) demonstrated that it was specifically limited to the skeletogenic PM of intact embryos. During the development of the PM, all five RNAs exhibited the same schedule of accumulation, appearing de novo, or increasing abruptly just before PM ingression, and remaining at relatively high levels thereafter. This pattern of RNA accumulation closely paralleled the pattern of synthesis of PM-specific proteins in general (Harkey and Whiteley, 1983) and of the SpLM 18-encoded protein specifically (Leaf et al., 1987). These results indicate that at least five distinct genes in the sea urchin, each of which encodes a PM-enriched or PM-specific mRNA, are expressed with tight coordination during development of the larval skeleton. They also demonstrate that expression of these genes in the PM is regulated primarily at the level of RNA abundance rather than RNA utilization.

Histone gene expression in interspecies hybrid echinoid embryos

Abstract The histones synthesized by three different interspecies hybrid echinoid embryos have been examined. In all three crosses, the species-specific F1 histone of the paternal parent is found in the chromatin of the hybrid. These findings provide direct evidence for the involvement of newly transcribed mRNA in the synthesis of this protein. F1 histone is a unique protein in that it is the only paternal protein detected in these hybrids. The possibility that the synthesis of histones is controlled differently from that of other proteins is discussed.

The interchangeability of stimulatory factors isolated from three microbial RNA polymerases

Abstract The DNA-dependent RNA polymerases of three bacteria were resolved by phosphocellulose chromatography into minimal RNA polymerases and stimulatory factors. The latter had no intrinsic RNA polymerase activity but promoted the synthesis of RNA by the minimal enzyme. Under some conditions, the factor from one species of bacteria was able to stimulate the minimal enzyme from a heterologous species.

The amino acid sequence of Micrococcusaerogenes rubredoxin

Abstract In the past few years, several non-heme iron proteins have been isolated and characterized (San Pietro, 1965) . Among others, the ferredoxins have come under extensive investigation. The amino acid sequences of Clostridium pasteurianum and C. butyricum ferredoxins have been reported from this laboratory ( Tanaka, et al., 1964 ; Benson, et al., 1966 ). Another non-heme iron protein, rubredoxin, was first obtained in the crystalline state from C. pasteurianum and was shown to replace ferredoxin in certain enzymatic reactions (Lovenberg and Sobel, 1965) . The protein was reported to contain one gram atom of non-heme iron per mole of protein and to have a molecular weight around 6,000. The present report is concerned with the determination of the complete amino acid sequence of M. aerogenes rubredoxin.

Bacteriophage SP82 induced modifications of Bacillus subtilis RNA polymerase result in the recognition of additional RNA synthesis initiation sites on phage DNA

Abstract The modification of the subunit structure of Bacillus subtilis RNA polymerase which occurs after infection with bacteriophage SP82 alters the transcriptional specificity of the enzyme. The data presented here determine which restriction endonuclease fragments of SP82 are used as initiation sites for RNA synthesis by the modified and unmodified enzymes. The analysis shows that the change in transcriptional specificity is due to the ability of the modified polymerase to initiate RNA synthesis at new sites on SP82 DNA. Reconstitution experiments using subunits isolated from the modified RNA polymerase indicate that the 28,000 molecular weight polypeptide coded by the phage is responsible for the change in initiation specificity.

A new polypeptide associated with RNA polymerase from Bacillus subtilis during late stages of vegetative growth

DNA-dependent RNA polymerase has been purified from Bacillus subtilis at various stages of vegetative cell growth. Polymerase isolated from cultures approaching the end of the logarithmic growth phase was associated with a 60,000-dalton polypeptide and was only 10–20% as active as polymerase isolated from rapidly growing cells. Appearance of this new polypeptide and the change in template activity occur prior to stage 0 of sporulation.

An increase in alkaline phosphatase in an in vitro system derived from Bacillus subtilis

Abstract There have been a number of reports that cell-free preparations from micro-organisms are capable of enzyme synthesis. Increases in the activity of tryptophan synthetase of Neurospora crassa ( Wainwright, 1959 ) and in the β-galactosidase of Escherichia coli ( Nisman and Fukuhara, 1959 ; Kameyama and Novelli, 1960 ) were the first to be described; more recently, the formation of tryptophan synthetase of E. coli ( Yura, et al., 1962 ) and amylase of Bacillus subtilis ( Oishi, et al., 1962 ) by particulate and soluble fractions have been reported. The present communication describes a similar observation with respect to alkaline phosphatase of B. subtilis and presents evidence concerning requirements and the effects of inhibitors. The requirement for a supernatant fraction from a genetically competent strain is discussed.

Subunit composition of Bacillus subtilis RNA polymerase during transcription

Abstract The subunit composition of RNA polymerase isolated from Bacillus subtilis was determined under conditions which allowed the formation of stable enzyme-DNA complexes and under conditions which permitted transcription. These investigations showed that the delta peptide was released upon formation of enzyme-DNA complexes whereas the sigma subunit was released during transcription.