[Developmental genetics by gene isolation: the dual 5S RNA gene system in Xenopus (author's transl)].
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Animals↗
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The developmentally regulated chorion gene families of insects.
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Animals↗
Altered expression of the developmentally regulated NFI gene family during phorbol ester-induced differentiation of human leukemic cells.
The nuclear factor I (NFI) family of transcription/replication proteins is required for the cell type-specific expression of a number of cellular and viral genes. There are four evolutionarily conserved NFI genes (NFI-A, -B, -C and -X) that encode DNA-binding proteins of similar or identical DNA-binding specificity. To address the role of this family of highly conserved transcription/replication proteins in cell differentiation and development, we have assessed the expression of NFI proteins (by DNA binding) and mRNAs of each of the four NFI genes during the in vitro differentiation of several human leukemic cell lines. These cell lines differ in their pattern of differentiation upon exposure to the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). In K562 and OCI/AML-3 cells, the levels of the four NFI mRNAs and the mobility of NFI-DNA complexes present in nuclear extracts changed during treatment with TPA. The TPA-induced change in mobility of NFI-DNA complexes in OCI/AML-3 cells was blocked by an inhibitor of protein synthesis, cycloheximide. These changes in NFI protein and mRNA expression appear to be cell type-or lineage-specific since no changes were seen during the TPA-induced differentiation of HL-60 or U937 cells. These studies indicate that although their DNA-binding specificities are similar, the proteins encoded by the four NFI genes may differ in their biological functions and play distinct roles in hematopoietic development.
Base Sequence↗
Expression of the pea S-adenosylmethionine decarboxylase gene is involved in developmental and environmental responses.
A cDNA, able to complement the S-adenosyl-L-methionine decarboxylase (SAMdC; EC 4.1.1.50)-defective yeast strain Y342, has been isolated from pea (Pisum sativum L.). Expression of the SAMdC gene was characterised during pea development. Northern analysis showed a differential expression of the pea SAMdC gene in vegetative and reproductive tissues. The highest SAMdC mRNA levels were found in undifferentiated callus and tissues with high rates of cell division, and at the onset of fruit development. SAMdC expression was also induced in senescing ovaries, probably in relation to an accumulation of spermine during ovary senescence. Finally, the levels of SAMdC transcripts in leaves and shoots were differentially affected by ozone treatment.
Adenosylmethionine Decarboxylase↗
Developmental expression of PC3 gene is correlated with neuronal cell birthday.
We examined the developmental expression of PC3, a nerve growth factor (NGF) early induced gene in PC12 cells, in the rat central nervous system (CNS) and we found that it represents a molecular marker of ongoing postmitotic neurons production. PC3 is initially expressed in the ventral quarter of the neural tube, at the level of the presumptive cervical spinal cord just where and when (10-11 days post coitum (dpc)) the motor neurons are arising. Subsequently, the appearance of PC3 expression follows a ventro-dorsal and a rostro-caudal gradient in the spinal cord and a caudo-rostral gradient across the brain vesicles that coincide, both spatially and temporally, with the gradients of neurogenesis described in the literature. As in PC12 cells, PC3 mRNA expression appears to be transient in vivo. In all regions of the CNS, it is restricted to the ventricular zone of the neuroepithelium, while neuronal precursors cease to express PC3 as they migrate to the mantle zone. Moreover, PC3 mRNA disappears from the various regions of the CNS as neurogenesis ceases.
Animals↗
Reactivation of Nedd-2, a developmentally down-regulated apoptotic gene, in apoptosis induced by a street strain of rabies virus.
A laboratory strain of rabies virus has been reported to induce apoptosis in experimental animals. The present study demonstrated that a bat strain and a primary canine rabies virus isolate also induced apoptosis in vivo. This death process involved reactivation of the caspase gene, Nedd-2, a developmentally down-regulated apoptotic gene. Expression of Nedd-2 was significantly up-regulated in infected adult and suckling mice. Reactivation of Nedd-2 in infected adult mice started at around day 3 and was prominent on day 5. The level of expression was constantly high up to the time that mice showed signs of illness. Expression of Nedd-2 correlated with the appearance of apoptotic nuclei within the infected brain, suggesting that reactivation of a developmentally down-regulated gene, Nedd-2, may be required for apoptotic elimination of cells damaged by infection.
Animals↗
A developmentally regulated membrane protein gene in Dictyostelium discoideum is also induced by heat shock and cold shock.
We have analyzed the expression of the Dictyostelium gene P8A7 which had been isolated as a cDNA clone from an early developmentally regulated gene. The single genomic copy generated two mRNAs which were subject to different control mechanisms: while one mRNA (P8A7S) was regulated like the cell-type-nonspecific late genes, the other one (P8A7L) was induced during development, when cells were allowed to attach to a substrate, and when cells were subjected to stress, such as heat shock and cadmium. Interestingly the same induction was also observed with cold shock. RNA processing was inhibited by heat and cold shock, leading to nuclear accumulation of a precursor. The translated region of the cDNA was common to both mRNAs and encoded an unusually hydrophobic peptide with the characteristics of a membrane protein.
Amino Acid Sequence↗
Proximal promoter binding protein contributes to developmental, tissue-restricted expression of the rat osteocalcin gene.
Osteocalcin is a 6 kD tissue-specific calcium binding protein associated with the bone extracellular matrix. The osteocalcin gene is developmentally expressed in postoproliferative rat osteoblasts with regulation at least in part at the transcriptional level. Multiple, basal promoter and enhancer elements which control transcriptional activity in response to physiological mediators, including steroid hormones, have been identified in the modularly organized osteocalcin gene promoter. The osteocalcin box (OC box) is a highly conserved basal regulatory element residing between nucleotides -99 and -76 of the proximal promoter. We recently established by in vivo competition analysis that protein interactions at the CCAAT motif, which is the central core of the rat OC box, are required for support of basal transcription [Heinrichs et al. J Cell Biochem 53:240-250, 1993]. In this study, by the combined utilization of electrophoretic mobility shift analysis, UV cross linking, and DNA affinity chromatography, we have identified a protein that binds to the rat OC box. Results are presented that support involvement of the OC box-binding protein in regulating selective expression of the osteocalcin gene during differentiation of the rat osteoblast phenotype and suggest that this protein is tissue restricted.
Animals↗
A gene expression screen in zebrafish embryogenesis.
A screen for developmentally regulated genes was conducted in the zebrafish, a system offering substantial advantages for the study of the molecular genetics of vertebrate embryogenesis. Clones from a normalized cDNA library from early somitogenesis stages were picked randomly and tested by high-throughput in situ hybridization for restricted expression in at least one of four stages of development. Among 2765 clones that were screened, a total of 347 genes with patterns judged to be restricted were selected. These clones were subjected to partial sequence analysis, allowing recognition of functional motifs in 163 among them. In addition, a portion of the clones were mapped with the aid of the LN54 radiation hybrid panel. The usefulness of the in situ hybridization screening approach is illustrated by describing several new markers for the characteristic structure in the fish embryo named the yolk syncytial layer, and for different regions of the developing brain.
Animals↗
Enhancer-binding proteins with a forkhead-associated domain and the sigma54 regulon in Myxococcus xanthus fruiting body development.
In response to starvation, Myxococcus xanthus initiates a developmental program that results in the formation of spore-filled, multicellular fruiting bodies. Many developmentally regulated genes in M. xanthus are transcribed from sigma(54) promoters, and these genes require enhancer-binding proteins. Here we report the finding of an unusual group of 12 genes encoding sigma(54)-dependent enhancer-binding proteins containing a forkhead-associated (FHA) domain as their N-terminal sensory domain. FHA domains in other proteins recognize phosphothreonine residues. An insertion mutation in one of these genes, Mx4885, caused a cell autonomous aggregation and sporulation defect. In-frame deletion mutants showed that the FHA domain is necessary for proper Mx4885 function. The altered pattern of developmental gene expression in the mutant implied that Mx4885 is on the pathway of response to the morphogenetic C-signal. Immunoblots specific for C-signal and FruA imply that the site of Mx4885 action is downstream of FruA synthesis on the C-signal transduction pathway. Mx4885 may help to coordinate the level of intracellular phosphorylated FruA (FruA-P) with the level of C-signal displayed on the signal donor cell. Because FHA domains respond to phosphothreonine-containing proteins, these results suggest a regulatory link to the abundant Ser/Thr protein kinases in M. xanthus.
Amino Acid Sequence↗
Distinct signaling pathways from the circadian clock participate in regulation of rhythmic conidiospore development in Neurospora crassa.
Several different environmental signals can induce asexual spore development (conidiation) and expression of developmentally regulated genes in Neurospora crassa. However, under constant conditions, where no environmental cues for conidiation are present, the endogenous circadian clock in N. crassa promotes daily rhythms in expression of known developmental genes and of conidiation. We anticipated that the same pathway of gene regulation would be followed during clock-controlled conidiation and environmental induction of conidiation and that the circadian clock would need only to control the initial developmental switch. Previous experiments showed that high-level developmental induction of the clock-controlled genes eas (ccg-2) and ccg-1 requires the developmental regulatory proteins FL and ACON-2, respectively, and normal developmental induction of fl mRNA expression requires ACON-2. We demonstrate that the circadian clock regulates rhythmic fl gene expression and that fl rhythmicity requires ACON-2. However, we find that clock regulation of eas (ccg-2) is normal in an fl mutant strain and ccg-1 expression is rhythmic in an acon-2 mutant strain. Together, these data point to the endogenous clock and the environment following separate pathways to regulate conidiation-specific gene expression.
Aerobiosis↗
flbD encodes a Myb-like DNA-binding protein that coordinates initiation of Aspergillus nidulans conidiophore development.
The timing of asexual fruiting body formation during Aspergillus nidulans colony development is precisely regulated so that conidiophores are typically produced 1-2 mm behind the growing edge of the colony. Mutations in any of four A. nidulans genes, flbB, flbC, flbD, or flbE, result in colonies that are delayed at least 24 hr in their ability to initiate conidiophore development resulting in fluffy colonies with conidiophores forming in the center, at least 12-15 mm behind the growing edge. The requirement for each of these four genes in determining the timing of developmental initiation precedes transcriptional activation of the primary developmental regulatory gene brlA, indicating a possible role for each gene in developmentally regulated activation of brlA expression. The wild-type flbD gene was isolated and shown to encode an approximately 1.6-kb mRNA that is present throughout the A. nidulans life cycle. The deduced FlbD protein sequence predicts a 314-amino-acid polypeptide with significant identity at its amino terminus to the DNA-binding domain of the Myb family of transcription factors indicating that FlbD probably functions as a sequence-specific transcriptional activator. Although conidiophore development does not normally occur in submerged culture, forced overexpression of flbD in submerged hyphae caused inappropriate activation of brlA expression and resulted in production of complex conidiophores that produced all of the distinct cell types observed in wild-type conidiophores including viable spores. This ability of flbD overexpression to activate conidiation requires brlA, flbB, and flbA (another early developmental regulator) but does not require flbC or flbE. We propose that FlbD functions during normal development by activating transcription of other genes required for development (such as brlA) and that FlbD activity is normally controlled post-transcriptionally by an unknown mechanism.
Aspergillus nidulans↗
Oligonucleotide-based strategies to reduce gene expression.
Research on embryonic development and differentiation provides a sensitive, but challenging opportunity to use a variety of techniques designed to modulate gene expression. Changes in the expression of a single gene can alter levels of other genes and provide information on developmentally regulated gene expression pathways. The morphological consequences of altered gene expression can link gene expression to developmental fate. Oligonucleotide-based approaches offer a variety of means to potentially disrupt normal gene expression. The basis for some of these approaches is presented in this review.
Animals↗
Tissue- and developmental stage-specific expression of the rat ornithine carbamoyltransferase gene in transgenic mice.
Rat ornithine carbamoyltransferase (OCT; EC 2.1.3.3) is encoded by a large gene of 75 kilobases. Expression of this gene is restricted to the liver and small intestine, and there is an increase in expression late in gestation. The recombinant gene carrying 1.3 kilobases of the 5' flanking region of the gene fused to the rat OCT cDNA was microinjected into fertilized eggs, and 17 transgenic mice were produced. Expression in the liver of the transgene was detected in three mice. In these mice, the transgene expression was observed exclusively in the liver and small intestine. Expression of the transgene in the intestine was comparable to that of the endogenous mouse OCT gene, whereas expression in the liver was much lower than that of the endogenous gene. The developmental pattern of expression of the transgene was similar to that of the endogenous gene. Therefore, the 5' flanking sequence of the rat OCT gene seems to be sufficient for the developmental and tissue-specific expression of the gene. An explanation for low expression in the liver remains the subject of ongoing study.
Animals↗
S-Phase progression mediates activation of a silenced gene in synthetic nuclei.
Aberrant expression of developmentally silenced genes, characteristic of tumor cells and regenerating tissue, is highly correlated with increased cell proliferation. By modeling this process in vitro in synthetic nuclei, we find that DNA replication leads to deregulation of established developmental expression patterns. Chromatin assembly in the presence of adult mouse liver nuclear extract mediates developmental stage-specific silencing of the tumor marker gene alpha-fetoprotein (AFP). Replication of silenced AFP chromatin in synthetic nuclei depletes sequence-specific transcription repressors, thereby disrupting developmentally regulated repression. Hepatoma-derived factors can target partial derepression of AFP, but full transcription activation requires DNA replication. Thus, unscheduled entry into S phase directly mediates activation of a developmentally silenced gene by (i) depleting developmental stage-specific transcription repressors and (ii) facilitating binding of transactivators.
Animals↗
Preferential rearrangement of developmentally regulated immunoglobulin VH1 genes in human B-lineage leukaemias.
Immunoglobulin heavy chain (IgH) variable region (VH) genes are rearranged and expressed in a programmed manner during B-cell development. In common with foetal/pre-immune B-cells, malignant B-lymphoid populations preferentially use a restricted repertoire of developmentally regulated VH genes. By nucleotide sequence analysis of polymerase chain reaction amplified IgH genes, we have compared the repertoire of VH1 family genes that are rearranged in mature, CD5+ B chronic lymphocytic leukaemia (CLL) with that in immature, CD5-B-lineage acute lymphoblastic leukaemia (ALL). The results revealed a non-random pattern pf VH1 usage in which no single VH1 family member was common to each of these disease groups. The VH1 gene, 51P1, which underlies an auto-antibody associated cross-reactive idiotype, 'G6', frequently expressed on foetal B-cells, was preferentially rearranged in CLL (three of nine rearranged alleles). Another developmentally regulated VH1 gene, 20P3, accounted for more than half of the VH1 specific IgH gene rearrangements in ALL (five of nine VH1 alleles). Such developmentally restricted VH1 genes may distinguish discrete, although not necessarily exclusive, stages or compartments in B-lymphopoiesis from which each of these disease types arise.
B-Lymphocytes↗
Involvement of differential gene expression and mRNA stability in the developmental regulation of the hsp 30 gene family in heat-shocked Xenopus laevis embryos.
Four complete hsp 30 genes have been isolated from Xenopus laevis: hsp 30A, hsp 30B (a pseudogene), hsp 30C, and hsp 30D. The hsp 30A and hsp 30C genes are first heat inducible at the early tailbud stage, as determined by RNase protection and RT-PCR assays. In this study, we determined by RT-PCR that the hsp 30D gene was first heat inducible (33 degrees C for 1 h) at the mid-tailbud stage, approximately 1 day later in development than hsp 30A and hsp 30C. Furthermore, using Northern blot analysis, we detected the presence of very low levels of hsp 30 mRNA at the heat-shocked late blastula stage. The relative levels of these pre-tailbud (PTB) hsp 30 mRNAs increased at the gastrula and neurula stage followed by a dramatic enhancement in heat shocked tailbud and tadpole stage embryos (50- to 100- fold relative to late blastula). Interestingly, treatment of blastula or gastrula embryos at high temperatures (37 degrees C for 1 h) or with the protein synthesis inhibitor, cycloheximide, followed by heat shock, led to enhanced accumulation of the pre-tailbud (PTB) hsp 30 mRNAs. hsp 70, hsp 87, and actin messages were not stabilized at high temperatures or by cycloheximide treatment. Finally, hsp 30D mRNA was not detected by RT-PCR analysis of cycloheximide-treated, heat-shocked blastula stage embryos, confirming that it is not a member of the PTB hsp 30 mRNAs. This study indicates that differential gene expression and mRNA stability are involved in the regulation of hsp 30 gene expression during early Xenopus laevis development.
Animals↗
The PAX gene egl-38 mediates developmental patterning in Caenorhabditis elegans.
Mutations in the C. elegans gene egl-38 result in a discrete set of defects in developmental pattern formation. In the developing egg-laying system of egl-38 mutant hermaphrodites, the identity of four uterine cells is disrupted and they adopt the fate of their neighbor cells. Likewise, the identity of two rectal epithelial cells in the male tail is disrupted and one of these cells adopts the fate of its neighbor cell. Genetic analysis suggests that the egl-38 functions in the tail and the egg-laying system are partially separable, as different egl-38 mutations can preferentially disrupt the different functions. We have cloned egl-38 and shown that it is a member of the PAX family of genes, which encode transcription factors implicated in a variety of developmental patterning events. The predicted EGL-38 protein is most similar to the mammalian class of proteins that includes PAX2, PAX5 and PAX8. The sequence of egl-38 mutant DNA indicates that the tissue-preferential defects of egl-38 mutations result from substitutions in the DNA-binding paired domain of the EGL-38 protein. egl-38 thus provides the first molecular genetic insight into two specific patterning events that occur during C. elegans development and also provides the opportunity to investigate the in vivo functions of this class of PAX proteins with single cell resolution.
Amino Acid Sequence↗