Prealbumin gene expression during mouse development studied by in situ hybridization

Localization of prealbumin mRNA in tissues from mice at various stages of gestation was investigated using in situ hybridization procedures. Prealbumin mRNA was detected as early as the 10th day of gestation. It was specifically localized in endodermal cells of the visceral yolk sac, tela choroidea, and hepatocytes. In the adult mice, prealbumin mRNA was localized in the hepatocytes and choroid plexus epithelial cells. These observations indicate that synthesis of prealbumin mRNA is initiated in several different types of cells at early stages of fetal development.     

Histone acetyltransferase activity during the cell cycle

Histone acetyltransferase activity was measured in isolated nuclei during the synchronous cell cycle of the myxomycete Physarum polycephalum. Nuclei were incubated with [14C]acetyl-coenzyme A and an excess of exogenous calf thymus histones. The activity is periodic during the cell cycle; it rises during the S-phase to reach a maximum in the early G2-period with a decline in mid and late G2. Comparison of the pattern of enzyme activity with the in vivo acetylation of histones during the cell cycle reveals that the enzyme activity does not wholly determine the acetylation state, indicating that other factors, including possibly the structural state of chromatin, are responsible for the observed cell cycle pattern of in vivo histone acetylation.     

Alpha 2-macroglobulin gene expression during rat development studied by in situ hybridization.

The sites of alpha 2-macroglobulin mRNA synthesis during rat development have been localized by in situ hybridization using a rat alpha 2-macroglobulin cDNA probe. Fetal liver was found to be the major site of alpha 2-macroglobulin mRNA synthesis. In addition, alpha 2-macroglobulin mRNA was detected in brain, spinal cord and eye. Alpha 2-Macroglobulin mRNA was quantitated by use of a sensitive RNAse protection assay. Maximal levels of alpha 2-macroglobulin mRNA were found in fetal livers shortly before birth. A rapid decline of alpha 2-macroglobulin mRNA occurred within 1 day after parturition. A similar time course, although at an approximately 20-fold lower level, was observed for alpha 2-macroglobulin mRNA in livers of pregnant rats. Alpha 2-Macroglobulin mRNA could also be detected in placenta. The levels were comparable to those found in maternal livers.     

Histone gene expression during sea urchin spermatogenesis: an in situ hybridization study

The expression of testis-specific and adult somatic histone genes in sea urchin testis was investigated by in situ hybridization. The testis-specific histone genes (Sp H2B-1 of Strongylocentrotus purpuratus and Sp H2B-2 of Lytechinus pictus) were expressed exclusively in a subset of male germ line cells. These cells are morphologically identical to replicating cells pulse-labelled with 3H-thymidine. Genes coding for histones expressed in adult somatic and late embryo cells (H2A-beta for S. purpuratus and H3-1 for L. pictus) were expressed in the same germ line cells, as well as in the supportive cells (nutritive phagocytes) of the gonad. All histone mRNAs detected in the male germ lineage declined precipitously by the early spermatid stage, before cytoplasmic reduction. The data suggest that both testis-specific and adult somatic histone genes are expressed in proliferating male germ line cells. Testis-specific gene expression is restricted to spermatogonia and premeiotic spermatids, but somatic histone expression is not. The decline of histone mRNA in nondividing spermatids is not merely a consequence of cytoplasmic shedding, but probably reflects mRNA turnover.     

REG gene expression in inflamed and healthy colon mucosa explored by in situ hybridisation

The regenerating islet-derived (REG) gene family encodes a group of proteins highly expressed in several human pathologies, many of which are associated with epithelial inflammation. All human family members, namely REG1A, REG1B, REG3A and REG4, are closely related in genomic sequence and all are part of the c-type lectin superfamily. REGs are highly expressed during inflammatory bowel disease (IBD)-related colonic inflammation and the in vivo expression pattern of REG1A and REG4 has been localised by using immunohistochemistry. However, the function of the encoded proteins is largely unknown and the cellular localisation of REG expression during colonic inflammation has not been described. Therefore, we have used in situ hybridisation to demonstrate the cellular localisation of REG expression in healthy and diseased colonic mucosa. Samples drawn from an IBD cohort including both inflamed and un-inflamed colonic mucosa are described, as are samples from non-IBD inflammation and healthy controls. Immunohistochemistry against known cell-type markers on serial sections has localised the expression of REGs to metaplastic Paneth cells (REG1A, REG1B and REG3A) and enteroendocrine cells (REG4), with a marked expansion of expression during inflammation. The group of REGs can, based on gene expression patterns, be divided into at least two groups; REG1A, REG1B and REG3A with their expression focused in the crypt base spreading from Paneth cells and REG4 being more highly expressed towards the luminal face. This exploration of expression pattern forms provides the background for further exploration of REG function in the intestine.     

Leu-callatostatin gene expression in the blowflies Calliphora vomitoria and Lucilia cuprina studied by in situ hybridisation: comparison with Leu-callatostatin confocal laser scanning immunocytochemistry

In situ hybridisation studies using a digoxigenin-labelled DNA probe encoding the Leu-callatostatin prohormone of the blowflies Calliphora vomitoria and Lucilia cuprina have revealed a variety of neurones in the brain and thoracico-abdominal ganglion, peripheral neurosecretory neurones, and endocrine cells of the midgut. With two exceptions, the hybridising cells are the same as those previously identified in immunocytochemical studies of sections and whole-mounts using Leu-callatostatin COOH-terminal-specific antisera. Within the brain and suboesophageal ganglion, there is a variety of neurones ranging from a single pair of large cells situated in the dorsal protocerebrum, to the several pairs of neurones in the tritocerebrum, some of which, in immunocytochemical preparations, can be seen to project via axons in the cervical connective to the thoracico-abdominal ganglion. In the medulla of the optic lobes, numerous small interneurones hybridise with the probe, as do clusters of similar-sized neurones close to the roots of the ocellar nerves. These results indicate that the Leu-callatostatin neuropeptides of the brain play a variety of roles in neurotransmission and neuromodulation. There are only three pairs of Leu-callatostatin-immunoreactive neurones in the thoracico-abdominal ganglion, at least two pairs of which project axons along the median abdominal nerve to provide extensive innervation of the hindgut. The Leu-callatostatin peripheral neurosecretory cells are located in close association with both nerve and muscle fibres in the thorax. In addition to neuronal Leu-callatostatin, the presence of the peptide and its mRNA has been demonstrated in endocrine cells in the posterior part of the midgut. These observations provide an example of a named brain/gut peptide in an insect.     

Characterisation of the nucleolar organising regions during the cell cycle in two varieties of Petunia hybrida as visualised by fluorescence in situ hybridisation and silver staining

The cell cycle-dependent spatial position, morphology and activity of the four nucleolar organising regions (NORs) of the Petunia hybrida cultivar Mitchell and the inbred line V26 have been analysed. Application of the silver staining technique and fluorescence in situ hybridisation on fixed root-tip material revealed that these interspecific hybrids possess four NORs of which only those of chromosome 2 are active during interphase, which implies that the NOR activity is not of parental origin. However, at the end of mitosis, activity of all NOR regions could be detected, suggesting that the high demand for ribosomes at this stage of the cell cycle requires temporal activity of all NORs. Using actin DNA probes as markers in fluorescence in situ hybridisation experiments enabled the identification of the individual petunia chromosomes. Received: 5 December 1997; in revised form: 2 March 1998 / Accepted: 2 March 1998     

Heat-shock gene expression and cell cycle changes during mammalian embryonic development

Synchronized regulation of cell division during gastrulation is essential for the regional proliferation of cells and pattern formation of the early CNS. The neural plate and neuroectoderm cells are a rapidly dividing and differentiating population of cells with a unique and rapid heat-shock response. Heat shock and the heat-shock genes were studied during neural plate development in a whole rat embryo culture system at 9.5-11.5 days. A lethal heat shock can cause cell death and severe developmental defects to the forebrain and eye during organogenesis. Heat shock can also result in acquired thermotolerance whereby cell progression is delayed at the G1/S and S/G2 boundaries of the cell cycle. This delay in cell cycle progression caused an overall lengthening of the cell cycle time of at least 2 hr. The heat shock genes may therefore function as cell cycle regulators in neuroectoderm induction and differentiation. The kinetics and expression of the hsp genes were examined in neuroectodermal cells by flow cytometry and Northern analysis. The levels of hsp mRNA 27, 71, 73, and 88 were identified following exposure at 42°C (nonlethal), 43deg;C (lethal) and 42deg;/43deg;C (thermotolerant) heat shock. Examination of hsp gene expression in the neural plate showed tight regulation in the cell cycle phases. Hsp 88 expression was enhanced at Go and hsp71 induction at G2 + M of the cell cycle. Cells exposed to a thermotolerant heat shock of 42deg;C induced hsp71 mRNA expression in all phases of the cell cycle with the mRNA levels of hsp27, 73, and 88 increased but relatively constant. Following a lethal heat shock, dramatic changes in hsp expression were seen especially enhanced hsp71 induction in late S phase. The regulated expression of hsps during the cell cycle at various phases could play a unique and important role in the fate and recovery of neuroectoderm cells during early mammalian embryo development. © 1993Wiley-Liss, Inc.     

Histone gene expression and chromatin structure in mammalian cell hybrids

DNA isolated from mammalian cell nuclear reveals discrete size patterns when partially digested with micrococcal nuclease. The DNA repeat lengths from different tissues within a species or from different species may vary. These differences have been attributed to the presence of different species of histone H1. To examine the nature of regulation of DNA repeat lengths and their possible relationship to histone H1, we have selected several mouse and human cell lines that differ in their DNA repeat lengths and examined them and their cell hybrids. 24 mouse X human and five mouse X mouse hybrid cell lines were analyzed. All the interspecific hybrids exhibited the repeat pattern characteristic of the murine parent. The mouse intraspecific hybrids had a repeat pattern of only one of the parents. We conclude that the partial human chromosome complements retained in the hybrids assume the repeat lengths exhibited by the mouse cells. Because H1 histones have been implicated in the determination of DNA repeat lengths, we also investigated the regulation of H1 histone expression in these cell hybrids. Purified H1 histones were radioactively labeled in vitro, and individual subfractions were subjected to proteolysis followed by gel electrophoresis. The resulting partial peptide maps off H1 histone subfractions A and B were distinguishable from one another and from different cell lines. In the mouse X human hybrids analyzed, only the mouse H1 histones were detected. These observations were extended to H2b by analysis of the hybrid cell histone by Triton-acid-urea gels. Neither the DNA repeat length nor histone expression is affected by the presence of any specific human chromosome. The fact that human genes are expressed in these hybrids suggests that the H1 histones of one species is able to interact with the chromatin of another species in a biologically funtional conformation. Analysis of the intraspecific PG19 X B82 (mouse X mouse) hybrids reveals the presence of H1 histone subfractions of the B82 mouse cells. Because these hybrids exhibit the nucleosome repeat length only of the PG19 cells, it appears that if histone H1 plays a role in determining the repeat length it does so in consort with other nonhistone chromosomal proteins.     

Histone H2B subtypes are dispensable during the yeast cell cycle

Saccharomyces cerevisiae contains two histone H2B protein subtypes, H2B1 and H2B2, which differ at 4 of 130 amino acids. We describe experiments that test whether both histone H2B subtypes are required for the completion of any stage in the yeast life cycle. Frameshift mutations were introduced into cloned copies of the H2B1 and H2B2 genes. These altered genes were integrated into the yeast genome by transformation and replaced the wild-type genes through recombination. We thus obtained strains that lacked functional H2B1 or H2B2 proteins. These mutant strains survive as haploids and homozygous diploids. During vegetative growth, they divide at the same rate as wild-type cells and are able to mate, sporulate and germinate. The h2b1^? cells grew more slowly after germination than h2b2^? or wild-type spores, but otherwise the mutants were indistinguishable from each other or from wild-type cells. We also attempted to make a strain that was mutant in both genes for H2B. We examined spores derived from a diploid that is heterozygous for both histone mutations. The two genes assort independently, so we expect one in four spores to be h2b1^?h2b2^?. Of 61 spore colonies examined, none was mutant at both loci. Our results indicate that the double mutant can germinate and bud once but cannot grow further. Since the yeast life cycle can be completed in the absence of either but not both histone H2B subtypes, we conclude that neither protein has a unique essential function.