Reinitiation Of Growth Research Articles

Chromatin remodeling by Pol II primes efficient Pol III transcription

The packaging of the genetic material into chromatin imposes the remodeling of this barrier to allow efficient transcription. RNA polymerase II activity is coupled with several histone modification complexes that enforce remodeling. How RNA polymerase III (Pol III) counteracts the inhibitory effect of chromatin is unknown. We report here a mechanism where RNA Polymerase II (Pol II) transcription is required to prime and maintain nucleosome depletion at Pol III loci and contributes to efficient Pol III recruitment upon re-initiation of growth from stationary phase in Fission yeast. The Pcr1 transcription factor participates in the recruitment of Pol II, which affects local histone occupancy through the associated SAGA complex and a Pol II phospho-S2 CTD / Mst2 pathway. These data expand the central role of Pol II in gene expression beyond mRNA synthesis.

Serum starvation-based method of ovarian cancer cell dormancy induction and termination in vitro.

Awakening and growth reinitiation by dormant cells may contribute to epithelial ovarian cancer (EOC) relapse. The links between these phenomena are loose because of the limited stock of compelling models of EOC dormancy. Here, we show a simple and convenient dormancy research protocol based on serum starvation. This study was conducted on established EOC cell lines A2780, OVCAR-3, and SKOV-3, as well as on primary EOC cells. Cell growth arrest and proliferation were monitored by assessing the Ki67 antigen, PKH26 fluorescence, and cell cycle distribution. In addition, cells were tested for ERK1/2/p38 MAPK activity ratio, apoptosis, and senescence. The study showed that 72-h serum starvation induces G0/G1 growth arrest of a significant fraction of cells, accompanied by reduced Ki67 and ERK1/2/p38 MAPK activity ratio, without signs of apoptosis or cellular senescence. Moreover, providing cells with 72 h of a medium enriched in 5% serum allows the culture to regain its proliferative potential. At the same time, we attempted to induce and terminate dormancy with Mitomycin C addition and withdrawal, which were unsuccessful. In conclusion, serum starvation is a convenient way to reliably induce dormancy in EOC cells, allowing them to be efficiently awakened for further mechanistic research in vitro.

Ribosomal RNA degradation induced by the bacterial RNA polymerase inhibitor rifampicin.

Rifampicin, a broad-spectrum antibiotic, inhibits bacterial RNA polymerase. Here we show that rifampicin treatment of Escherichia coli results in a 50% decrease in cell size due to a terminal cell division. This decrease is a consequence of inhibition of transcription as evidenced by an isogenic rifampicin-resistant strain. There is also a 50% decrease in total RNA due mostly to a 90% decrease in 23S and 16S rRNA levels. Control experiments showed this decrease is not an artifact of our RNA purification protocol and therefore due to degradation in vivo. Since chromosome replication continues after rifampicin treatment, ribonucleotides from rRNA degradation could be recycled for DNA synthesis. Rifampicin-induced rRNA degradation occurs under different growth conditions and in different strain backgrounds. However, rRNA degradation is never complete, thus permitting the reinitiation of growth after removal of rifampicin. The orderly shutdown of growth under conditions where the induction of stress genes is blocked by rifampicin is noteworthy. Inhibition of protein synthesis by chloramphenicol resulted in a partial decrease in 23S and 16S rRNA levels whereas kasugamycin treatment had no effect. Analysis of temperature-sensitive mutant strains implicate RNase E, PNPase, and RNase R in rifampicin-induced rRNA degradation. We cannot distinguish between a direct role for RNase E in rRNA degradation versus an indirect role involving a slowdown of mRNA degradation. Since mRNA and rRNA appear to be degraded by the same ribonucleases, competition by rRNA is likely to result in slower mRNA degradation rates in the presence of rifampicin than under normal growth conditions.

Cytosolic free calcium dynamics as related to hyphal and colony growth in the filamentous fungal pathogen Colletotrichum graminicola

Tip growth of pollen tubes and root hairs of plants is oscillatory and orchestrated by tip-focussed variations of cytosolic free calcium ([Ca2+]cyt). Hyphae of filamentous fungi are also tubular tip-growing cells, and components of the Ca2+ signalling machinery, such as Ca2+ channels and Ca2+ sensors, are known to be important for fungal growth. In this study, we addressed the questions if tip-focussed [Ca2+]cyt transients govern hyphal and whole-colony growth in the maize pathogen Colletotrichum graminicola, and whether colony-wide [Ca2+]cyt dynamics rely on external Ca2+ or internal Ca2+ stores. Ratiometric fluorescence microscopy of individual hyphae expressing the Ca2+ reporter Yellow Cameleon 3.6 revealed that Ca2+ spikes in hyphal tips precede the re-initiation of growth after wounding. Tip-focussed [Ca2+]cyt spikes were also observed in undisturbed growing hyphae. They occurred not regularly and at a higher rate in hyphae growing at a medium-glass interface than in those growing on an agar surface. Hyphal tip growth was non-pulsatile, and growth speed was not correlated with the rate of spike occurrence. A possible relationship of [Ca2+]cyt spike generation and growth of whole colonies was assessed by using a codon-optimized version of the luminescent Ca2+ reporter Aequorin. Depletion of extracellular free Ca2+ abolished [Ca2+]cyt spikes nearly completely, but had only a modest effect on colony growth. In a pharmacological survey, some inhibitors targeting Ca2+ influx or release from internal stores repressed growth strongly. However, although some of those inhibitors also affected [Ca2+]cyt spike generation, the effects on both parameters were not correlated. Collectively, the results indicate that tip growth of C. graminicola is non-pulsatile and not mechanistically linked to tip-focused or global [Ca2+]cyt spikes, which are likely a response to micro-environmental parameters, such as the physical properties of the growth surface.

Isolation, purification, and culture of oogonial stem cells from adult human and bovine ovarian cortex

BackgroundThe longstanding belief that women are born with a finite ovarian reserve has been debated for almost a decade, ever since the discovery, and subsequent isolation, of purported oogonial stem cells (OSCs). These cells, described in both mice and man, seemingly have the ability to generate oocytes when cultured in vitro under specific conditions, resulting in live offspring in the case of mice. The aims of this study were to verify independently the existence of OSCs in human ovarian tissue and to determine whether they could be isolated from bovine tissue. MethodsWe isolated OSCs from disaggregated bovine and human ovarian cortex using a fluorescence-activated cell sorting (FACS)-based technique, with cells being sorted for the presence of VASA, a germ-cell-specific protein. The cells were subsequently cultured in vitro without the need for a feeder layer, and mRNA expression of germline-specific markers was analysed with RT-PCR. The cells were transduced to express green fluorescent protein (GFP) by use of a lentivirus for the purposes of tracking. FindingsA rare population of mitotically active VASA-positive cells was isolated from both bovine and human tissue and they have been grown in vitro for several months. Up to November, 2013, cells were retrieved from three human samples, but isolation has been replicated on five separate occasions in bovine ovary samples. RT-PCR demonstrated consistent expression of several germline markers, including IFITM3, DPPA3, PRDM1, POU5F1, and KIT. It has been possible to cryopreserve the cells with successful re-initiation of growth on thawing, and stable expression of GFP was achieved after lentivirus transduction. InterpretationThe expression of germline markers indicates that these cells have characteristics of germline (oogonial) stem cells. To our knowledge, this is the first report of such cells being isolated from bovine ovarian cortex and corroborates a previous report showing the isolation of OSCs from human ovary. These cells provide a novel model for investigating germ-cell development; future experiments will involve injection of OSCs into ovarian cortex to assess whether they have the capability to undergo follicle formation and oocyte development in an in-vitro environment. If their potential can be harnessed, OSCs might have a future role in clinical applications—eg, in fertility preservation. FundingUK Medical Research Council.

Genetic and Nongenetic Determinants of Cell Growth Variation Assessed by High-Throughput Microscopy

In microbial populations, growth initiation and proliferation rates are major components of fitness and therefore likely targets of selection. We used a high-throughput microscopy assay, which enables simultaneous analysis of tens of thousands of microcolonies, to determine the sources and extent of growth rate variation in the budding yeast (Saccharomyces cerevisiae) in different glucose environments. We find that cell growth rates are regulated by the extracellular concentration of glucose as proposed by Monod (1949), but that significant heterogeneity in growth rates is observed among genetically identical individuals within an environment. Yeast strains isolated from different geographic locations and habitats differ in their growth rate responses to different glucose concentrations. Inheritance patterns suggest that the genetic determinants of growth rates in different glucose concentrations are distinct. In addition, we identified genotypes that differ in the extent of variation in growth rate within an environment despite nearly identical mean growth rates, providing evidence that alleles controlling phenotypic variability segregate in yeast populations. We find that the time to reinitiation of growth (lag) is negatively correlated with growth rate, yet this relationship is strain-dependent. Between environments, the respirative activity of individual cells negatively correlates with glucose abundance and growth rate, but within an environment respirative activity and growth rate show a positive correlation, which we propose reflects differences in protein expression capacity. Our study quantifies the sources of genetic and nongenetic variation in cell growth rates in different glucose environments with unprecedented precision, facilitating their molecular genetic dissection.

Raf-induced MMP9 disrupts tissue architecture of human breast cells in three-dimensional culture and is necessary for tumor growth in vivo.

Organization into polarized three-dimensional structures defines whether epithelial cells are normal or malignant. In a model of morphogenesis, we show that inhibiting key signaling pathways in human breast cancer cells leads to "phenotypic reversion" of the malignant cells. Using architecture as an endpoint, we report that, in all cases, signaling through Raf/MEK/ERK disrupted tissue polarity via matrix metalloproteinase9 (MMP9) activity. Induction of Raf or activation of an engineered, functionally inducible MMP9 in nonmalignant cells led to loss of tissue polarity, and reinitiated proliferation. Conversely, inhibition of Raf or MMP9 with small molecule inhibitors or shRNAs restored the ability of cancer cells to form polarized quiescent structures. Silencing MMP9 expression also reduced tumor growth dramatically in a murine xenograft model. LC-MS/MS analysis comparing conditioned medium from nonmalignant cells with or without active MMP9 revealed laminin 111 (LM1) as an important target of MMP9. LM1 has been implicated in acinar morphogenesis; thus, its degradation by MMP9 provides a mechanism for loss of tissue polarity and reinitiation of growth associated with MMP9 activity. These findings underscore the importance of the dynamic reciprocity between the extracellular matrix integrity, tissue polarity, and Raf/MEK/ERK and MMP9 activities, providing an axis for either tissue homeostasis or malignant progression.

Inositol phosphoceramide synthase is a regulator of intracellular levels of diacylglycerol and ceramide during the G1 to S transition in Saccharomyces cerevisiae

We recently reported that DAG (diacylglycerol) generated during sphingomyelin synthesis plays an important role in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells [Cerbon and Lopez-Sanchez (2003) Biochem. J. 373, 917-924]. In yeast cells, IPC (inositol phosphoceramide) synthase catalyses the transfer of phosphoinositol from phosphatidylinositol to ceramide to form IPC and generates DAG. In the present study, we found that, during the G1 to S transition after N2-starvation, there was a significant increase in the synthesis of IPC accompanied by a progressive increase (up to 6-fold) in the level of DAG. The increased DAG levels coincided with decrements in ceramide and sphingoid base levels, conditions that are adequate for the activation of putative protein kinase C required for the G1 to S transition and proliferation of yeast cells. To separate the role of DAG generated during IPC synthesis from that originating from other sources, we utilized beta-chloroalanine and myriocin, inhibitors of serine:palmitoyl-CoA transferase, the first committed step in sphingolipid synthesis, to avoid accumulation of sphingolipid intermediates. When the synthesis of sphingolipids was inhibited, DAG accumulation was significantly decreased and the G1 to S transition was blocked; such blockage was avoided by metabolic complementation with phytosphingosine. The DAG/ceramide ratio was 0.27 and it changed to 2.0 during growth re-initiation, suggesting that the synthesis of phosphosphingolipids could act to switch growth arrest (increased ceramide) to a mitogenic signal (increased DAG), and that this signalling process is preserved in yeast and mammalian cells.

Inactivation of gacS does not affect the competitiveness of Pseudomonas chlororaphis in the Arabidopsis thaliana rhizosphere.

Quorum-sensing-controlled processes are considered to be important for the competitiveness of microorganisms in the rhizosphere. They affect cell-cell communication, biofilm formation, and antibiotic production, and the GacS-GacA two-component system plays a role as a key regulator. In spite of the importance of this system for the regulation of various processes, strains with a Gac(-) phenotype are readily recovered from natural habitats. To analyze the influence of quorum sensing and the influence of the production of the antibiotic phenazine-1-carboxamide on rhizosphere colonization by Pseudomonas chlororaphis, a gnotobiotic system based on Arabidopsis thaliana seedlings in soil was investigated. Transposon insertion mutants of P. chlororaphis isolate SPR044 carrying insertions in different genes required for the production of N-acyl-homoserine lactones and phenazine-1-carboxamide were generated. Analysis of solitary rhizosphere colonization revealed that after prolonged growth, the population of the wild type was significantly larger than that of the homoserine lactone-negative gacS mutant and that of a phenazine-1-carboxamide-overproducing strain. In cocultivation experiments, however, the population size of the gacS mutant was similar to that of the wild type after extended growth in the rhizosphere. A detailed analysis of growth kinetics was performed to explain this phenomenon. After cells grown to the stationary phase were transferred to fresh medium, the gacS mutant had a reduced lag phase, and production of the stationary-phase-specific sigma factor RpoS was strongly reduced. This may provide a relative competitive advantage in cocultures with other bacteria, because it permits faster reinitiation of growth after a change to nutrient-rich conditions. In addition, delayed entry into the stationary phase may allow more efficient nutrient utilization. Thus, GacS-GacA-regulated processes are not absolutely required for efficient rhizosphere colonization in populations containing the wild type and Gac(-) mutants.

Growth Kinetics of Salmonella Isolates in a Laboratory Medium as Affected by Isolate and Holding Temperature

Salmonella isolates were surveyed for their growth kinetics in a laboratory medium for the purpose of identifying isolates suitable for modeling experiments. In addition, the effect of holding stationary phase Salmonella cultures at different temperatures on their subsequent growth kinetics was evaluated for the purpose of developing a protocol to prevent the need for midnight sampling in modeling experiments. In Experiment 1, 16 isolates of Salmonella, 2 from the American Type Culture Collection (ATCC) and 14 from broiler operations, were surveyed for their growth kinetics in brain heart infusion (BHI) broth at 40°C. Lag time (P = 0.005) and growth rate (P = 0.022) were affected by identity of the isolate. Lag time ranged from 0.73 to 1.38 h, whereas growth rate ranged from 0.78 to 0.94 log10 CFU/ml/h. Overall, isolate S1 (Salmonella infantis from ATCC) was the fastest growing. In Experiment 2, 4 isolates of Salmonella, 1 from ATCC and 3 from broiler operations, were used to determine whether holding temperature influences subsequent growth kinetics. Salmonella isolates were grown to stationary phase at 37°C in BHI and then held for 24 h at 5, 22, or 37°C before dilution and reinitiation of growth in BHI at 37°C. Holding temperature did not alter or interact with identity of the isolate to alter subsequent growth kinetics. From the latter finding, a protocol was devised in which a dual-flask system is used to prevent the need for midnight sampling in modeling experiments. Similar to the results obtained in Experiment 1, identity of the isolate had only minor effects on growth kinetics in Experiment 2 indicating that all isolates examined were suitable for modeling experiments.

Polyamines in normal and auxin‐induced strawberry fruit development

The possible involvement of polyamines during strawberry (Fragaria×ananassa Duch.) fruit development was investigated. Putrescine, spermidine, and spermine were identified in strawberry receptacles and achenes at all stages of development. Total (free) polyamine levels decreased from a maximum of 485 nmol g−1 fresh weight at pollination to a minimum of 55 nmol g−1 fresh weight in ripe receptacles. Total polyamine concentrations during corresponding stages of development were consistently higher in achenes than in receptacles, and ranged from 891 to 203 nmol g−1 fresh weight. Removal of achenes from the surface of developing receptacles 10 days after pollination reduced receptacle growth, and re‐initiation of growth by application of 1 mMα‐naphtaleneacetic acid (α‐NAA) was accompanied by a rapid increase in polyamine concentrations 24 h after treatment. Polyamine content per receptacle increased >3‐fold in normally developing receptacles and in de‐achened, auxin‐treated receptacles 10 days after removal of achenes, but did not increase during this period in de‐achened receptacles not treated with exogenous auxin. α‐NAA increased growth and polyamine levels to a greater extent than the structurally related, but less effective auxin, β‐NAA. Polyamine concentrations in receptacles with intact achenes remained similar to those of auxin depleted (de‐achened) receptacles, implying that the concentration of these compounds may not be limiting following achene removal.

Why Do Many Ruminal Bacteria Die and Lyse So Quickly?

Studies using 15N have indicated that as much as 50% of the microbial mass turns over before N passes to the lower gut, and this N recycling significantly decreases the availability of microbial protein. Protozoa digest bacteria and smaller protozoa, but bacterial protein can turn over even if protozoa are not present. Fibrobacter succinogenes cultures lyse even when they are growing, and the lysis rate is independent of growth rate. When extracellular sugar is depleted, F. succinogenes secretes an extracellular proteinase that inactivates the autolysins. This method of autolytic regulation decreases the turnover of stationary cells. Bacteriophage and anaeroplasma can cause lysogeny, but, as yet, there is little proof that these processes are important determinants of bacterial turnover in vivo. Dietary manipulations (e.g., salt feeding and particle size reduction) that increase liquid and solid dilution rates can increase bacterial flow by decreasing bacterial residence time and turnover. Some dead ruminal bacteria are able to maintain their cellular integrity, and the ratio of dead to live cells in ruminal fluid may be as great as 10:1. Bacterial survival appears to be at least partially explained by the method of sugar transport. When bacteria rely solely on mechanisms of ion-coupled sugar symport, an energized membrane is necessary for the reinitiation of growth. If group translocation (phosphotransferase system) is the mechanisms of transport., uptake can be driven by phosphoenolpyruvate, and an energized membrane and the storage of intracellular reserve materials are not an absolute criteria for survival. In some cases, N deprivation accelerates death. When Prevotella ruminicola was limited for N under conditions of excess energy, methylglyoxal production caused a rapid decrease in viability. The impact of bacterial death in the rumen is not clear-cut. If the rate of fermentation is zero-order with respect to cell concentration (substrate-limited), cell death would have little impact on digestion.

The influence of environmental factors on the growth of Sphaerechinus granularis (Lamarck) (Echinodermata: Echinoidea)

The main growth period of the sea urchin Sphaerechinus granularis (Lamarck) in Brittany occurs in summer and autumn with a faster growth rate in the juveniles. The growth curve was similar in laboratory and field populations except during the winter phase where growth rate decreased in the field but was negative from the second year-class in laboratory conditions. To determine the factors which regulate somatic growth, the seasonal variations in feeding, gonadal growth and temperature were analysed. The ingestion rate of laboratory urchins and the repletion index of the natural population were low from December to April and at the end of summer irrespective of the age of the sea urchins. The changes however were less obvious in the juveniles. In the adults, the gonad index increased from February to April before spawning. The decrease in the growth rate in October was influenced more by falling sea temperature than by a change in the feeding rate which decreased a month later. Temperature is not the only factor involved in the reinitiation of growth, in April. This coincides with the end of spawning and when the feeding activity recommenced. The resumption of feeding is influenced by the temperature as indicated by a significant correlation between the feeding indices and the seawater temperature one month before.

Banks of algal microscopic forms: hypotheses on their functioning and comparisons with seed banks

It has been proposed that a 'bank of microscopic forms' functions as a survival mechanism for benthc macroalgae, m some ways analogous to the 'seed bank' of terrestrial plants. Four aspects related to this hypothesis are examined here, namely (1) origin and morphology of the structures forming banks, (2) their specific compositions, (3) environmental conditions that allow their survival in a state of suspended growth, and conditions that trigger reinitiation of growth; and (4) losses from the banks by diverse causes Although the structures at which growth suspension occurs are of very different morphologies, and probably are also different in their capacities of temporal displacement, both types of banks are regulated by similar environmental factors and subjected to similar types of constraints. Some important differences between the 2 banks are likely to stem from the lesser longevity of the algal microscopic stages, which would limit the duration of banks of microscopic forms

Transcriptional activities in dry seed nuclei indicate the timing of the transition from embryogeny to germination.

To investigate processes regulating the reinitiation of growth and differentiation during seed germination, we have studied the transcriptional activities of sets of genes that are expressed at specific stages of embryogeny and post-germination in the higher plant Brassica napus L. We show that transcripts from a subset of the genes are elongated in nuclei isolated from dry seeds, indicating that these genes are competent to be transcribed in desiccated and quiescent mature embryos. Analysis of the specific transcripts produced in dry seed nuclei indicates that the changes in gene expression patterns associated with germination are not initiated during late embryogeny. The results suggest that the transition from an embryonic to a postgerminative program of development occurs after seeds are rehydrated.

The role of FIS in trans activation of stable RNA operons of E. coli.

The thrU(tufB) operon of Escherichia coli is endowed with a cis-acting region upstream of the promoter, designated UAS for Upstream Activator Sequence. A protein fraction has been isolated that binds specifically to DNA fragments of the UAS, thus forming three protein-DNA complexes corresponding to three binding sites on the UAS. It stimulates in vitro transcription of the operon by facilitating the binding of the RNA polymerase to the promoter. All three protein-DNA complexes contain one and the same protein. Dissociation constants for the three complexes have been determined, the lowest being in the sub-nanomolar range. The protein also binds to the UAS of the tyrT operon and to the UAS upstream of the P1 promoter of the rrnB operon, suggesting that transcription of the three operons, if not of more stable RNA operons, is activated by a common trans activator. We demonstrate that the E.coli protein FIS (Factor for Inversion Stimulation) also binds to the UAS of the thrU(tufB) operon forming three protein-DNA complexes. A burst of UAS- and FIS-dependent promoter activity is observed after reinitiation of growth of stationary cultures in fresh medium.

Transforming Growth Factor-α Stimulates Proto-Oncogene c-junExpression and a Mitogenic Program in Primary Cultures of Adult Rat Hepatocytes

Human transforming growth factor-alpha (TGF-alpha, MW 5547) initiates a mitogenic program in "quiescent" 11-to 13-day-old primary cultures of adult rat hepatocytes. Using validated growth reinitiation assays and chemically defined conditions (Koch and Leffert, 1979a) that simulate proto-oncogene expression in regenerating liver (Kruijer et al., 1986), we find that 5.4 nM TGF-alpha stimulates: (i) increases in rates of amiloride-sensitive 22Na+ uptake; (ii) a transient induction in steady-state mRNA levels of proto-oncogene c-jun; (iii) specific increases in hepatocyte nuclear [3H]dT labeling indices, augmented synergistically by insulin and glucagon; and (iv) increases in rates of S-phase entry. Comparative studies indicate that TGF-alpha is a more effective hepatocyte growth promoter than mouse epidermal growth factor. These observations, and published reports linking normal and cancerous liver as biosynthetic sources of TGF-alpha, suggest an autocrine or paracrine role for TGF-alpha in the control of hepatic growth, regeneration, and gene expression.

Storage of the phytochrome-mediated phototropic stimulus of moss protonemal tip cells

A phytochrome-regulated phototropic response of the moss Ceratodon purpureus was investigated. Chlorotetracycline (CTC) was used to visualize membrane-associated calcium gradients in the tip cell of moss caulonemal filaments. A tip-to-base Ca(2+) gradient was observed. The ionophore monensin rapidly inhibited the growth of the tip cell and abolished the CTC fluorescence. Six hours after transferring to inhibitor-free medium, protonemal growth resumed and reached the normal growth rate within 12 h. The growth was accompanied by a reappearance of the CTC-fluorescence gradient. Unilateral irradiation given during the monensin treatment or after the treatment during the period when growth inhibition persisted led, with the re-initiation of growth, to a typical positive phototropic bending in complete darkness. Far-red light applied just before the growth response started, or during growth inhibition, abolished the phototropic response. The phytochrome-mediated signal was qualitatively (position) and quantitatively (degree of bending) memorized. Signal perception and response could be separated temporally. This result indicates that at least under some circumstances, e.g. under the influence of monensin, the phytochrome-mediated signal can be stored for several hours in darkness. Calcium seems to be essential for the processing of polar growth only. A specific function (second messenger) in phytochrome-dependent signal transduction could not be confirmed.

Anatomical Changes During Ontogeny of the Peanut (Arachis hypogaea L.) Fruit: Mature Megagametophyte through Heart-Shaped Embryo

Correlative light (LM) and scanning electron microscopy (SEM) of the same microtomed section was used to investigate components within the embryo sac of Arachis to the heart-shaped embryo phase of development. Morphometric data on the locule, ovule, embryo sac, and proembryo show that they reach maximum size during aerial development ca. 5 days after anthesis. After the onset of peg elongation, these components contract in size, and the apical locule and its components contract more than the basal. At the time of maximum aerial size, the proembryo is in the eight-cell stage and remains at that stage until the peg tip penetrates the soil surface. The first observed changes in the proembryo on reinitiation of growth is the swelling of the two basal tiers of cells from which the suspensor forms. Correlative LM and SEM of endosperm show physical contacts, in the form of cytoplasmic strands, between the connective cell layer at the base of the proembryo and the free-nuclear endosperm. These cytoplasmic strands are also layered against the integumentary tapetum and extend to the chalazal end of the embryo sac. These connections may have a role in the supply of nutrients to the developing embryo.

Growth defects in the chloride and MOCVD VPE of lattice matched layers of Ga0.47In0.53As on InP

A nominally composition matched MOCVD grown layer of Ga0.47In0.53As deposited on a “chloride” grown substrate was examined in section by TEM. The anomalous weak contrast defects commonly seen in VPE deposited alloy material are characterized and their origin in valley growth discussed. The interface region between the “chloride” grown substrate and MOCVD overgrowth is also described and the dislocation microstructure observed there interpreted as a function of the misfits which can arise through the critical reinitiation of growth having an incorrect composition when the active gas flows are adjusted at too low a temperature.