Cell-cell interactions during plant development.
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Cell Communication↗
Biomedical subjects
B R Char
Publications and source records attributed to B R Char.
Homeobox genes in the functioning of plant meristems.
The maize homebox gene knotted1 (kn1) is expressed in vegetative and floral meristems and is down-regulated at the site of primordia formation. kn1-related genes from maize and other species also show meristem-specific expression and offer additional tools for studying the activities of shoot meristems. Members of this gene family are expressed early in embryogenesis, providing molecular markers for meristem initiation. Ectopic expression of either kn1 or a related Arabidopsis gene, KNAT1, causes dramatic alterations in Arabidopsis and tobacco leaf morphology. Most significantly, meristems form on the leaf, producing small shoots. We discuss whether the phenotypes can be interpreted as changes in positional information or timing of determination.
Gene Expression Regulation, Plant↗
A POU gene required for early cleavage and protein accumulation in the sea urchin embryo.
SpOct is a POU gene expressed during oogenesis and early embryogenesis of the sea urchin, Strongylocentrotus purpuratus. In the first use of antisense technology in the sea urchin embryo, we report that disruption of SpOct gene function in 1-cell zygotes by the injection of antisense oligodeoxynucleotides arrests development prior to the first cell division. We show that single-stranded antisense oligodeoxynucleotides specifically block cleavage, and that injection of SpOct mRNA overcomes this block. The accumulation of [35S]methionine into zygotically synthesized protein is significantly reduced in antisense-injected embryos. DNA synthesis is also reduced by the antisense regimen as expected from the antisense inhibition of protein accumulation. That protein accumulation prior to the first cleavage is retarded by antisense targeting of a transcription factor is very surprising in light of classical work showing that the activation of protein synthesis does not require zygotic transcription. We conclude that either some new transcription is obligate for the accumulation of new protein, or that the SpOct gene plays a novel, non-transcriptional role in this process.
Animals↗
SpOct, a gene encoding the major octamer-binding protein in sea urchin embryos: expression profile, evolutionary relationships, and DNA binding of expressed protein.
We have characterized a sea urchin gene, SpOct, that encodes a 78-kDa POU-domain protein related to mammalian Oct-1 and Oct-2. The SpOct protein binds octamer elements in the promoters of the alpha H2B (Bell et al., 1992, Dev. Biol. 150, 363-371) and CyIIIa actin genes, and it closely resembles the major octamer-binding activity obtained from sea urchin blastula nuclear lysates in the size of its DNase I footprint on a canonical octamer element and in its relative binding affinity (Kr) for the octamer element versus poly(dAT) (1.4 x 10(4)). Moreover, partial protein sequences obtained from affinity-purified octamer-binding protein match sequences present in SpOct. These data suggest that SpOct is closely related to, if not identical with, the major octamer-binding activity in blastula nuclear extracts. RNA gel blots reveal four forms of SpOct mRNA, ranging in size from 4 to 12 kb. They are regulated coordinately in the embryo: all are present in the unfertilized egg, increase 28-fold in amount by the 8-hr blastula stage, and decline 6-fold by the 12-hr blastula stage. The same four size classes of SpOct mRNAs are present in several adult tissues, although their relative amounts vary. The temporal profile of SpOct mRNA expression in embryos closely resembles that of the alpha histone H2B gene. Our previous work (Bell et al., 1992) showed that expression of the alpha H2B gene in blastula-stage embryos was entirely dependent on an octamer element. Together, these data strongly suggest that SpOct may be the key regulator of the alpha H2B gene.
Amino Acid Sequence↗
An octamer element is required for the expression of the alpha H2B histone gene during the early development of the sea urchin.
Early (alpha) histone genes are one of several histone gene families in the sea urchin genome. They are expressed at high levels in blastula-stage embryos and are inactivated by the early gastrula stage. By microinjecting mutant early H2B genes into sea urchin zygotes and monitoring their transcriptional activity in blastula- and gastrula-stage embryos, we sought to identify the cis-regulatory elements responsible for this dramatic change in early H2B gene activity. We found that deletion of DNA 5' of -71 and 3' of +591 did not affect the timing or magnitude of early H2B gene expression. Neither was early H2B gene expression affected by the replacement of sequences downstream of -36 with the corresponding region of the L1 late H2B gene, expressed after the peak transcription of the early H2B gene. Further deletion of early H2B promoter sequences from -71 to -56, removing a conserved octamer element, resulted in near-complete inactivation of the early H2B gene in both blastula- and gastrula-stage embryos. Also inactivating early H2B gene expression were an internal deletion of the octamer element and a base substitution mutation that altered its sequence. This base substitution mutation also caused a parallel reduction in the ability of the octamer element to bind a factor present in nuclear extracts of sea urchin blastulae. These data strongly suggest that the proper expression of the early H2B gene in cleavage- and blastula-stage embryos depends on the octamer element and a factor with which it interacts.
Animals↗
Activation of a late H2B histone gene in blastula-stage sea urchin embryos by an unusual enhancer element located 3' of the gene.
In the sea urchin embryo, late histone genes are transcribed at low levels during cleavage and blastula formation and at substantially higher levels in later stages of embryogenesis. To investigate the molecular basis of the stage-specific expression of a late H2B histone gene, we injected mutant genes lacking portions of 5'- and 3'-flanking regions into Lytechinus pictus embryos and monitored their expression by RNase protection. A 200-bp region located 489 bp downstream of the mRNA 3' terminus was necessary for the increase in transcription of the late H2B gene at the mid-blastula stage of development. DNase I and methylation interference footprint analyses located only one factor-binding site in this region, and gel mobility shift experiments showed that the DNA-binding activity of this factor (designated H2B abp 1) paralleled the transcriptional activity of the L1 H2B gene. Additional mutagenesis and microinjection experiments located the activator element to a 32-bp DNA segment that includes the H2B abp 1-binding site. These experiments also showed that the 32-bp fragment functions independently of position and orientation and therefore has the hallmarks of an enhancer. That this fragment contains most or all of the L1 H2B gene transcription-stimulatory activity makes it unusual among enhancerlike elements, which generally consist of several clustered factor-binding sites that act additively or cooperatively to affect transcription. The nucleotide sequence of the L1 H2B enhancer element suggests that the trans-acting factor that interacts with it is a member of the antennapedia or engrailed class of homeodomain proteins.
Animals↗