Caulobacter crescentus carries a flagellum and is motile only during a limited time in its cell cycle. We have asked if the biochemical machinery that mediates chemotaxis exists coincident with the cell's structural ability to respond to a chemotactic signal. We first demonstrated that one function of the chemotaxis machinery, the ability to methylate the carboxyl side chains of a specific set of membrane proteins (methyl-accepting chemotaxis proteins, MCPs), is present in C. crescentus. This conclusion is based on the observations that (i) methionine auxotrophs starved of methionine can swim only in the forward direction (comparable to smooth swimming in the enteric bacteria), (ii) a specific set of membrane proteins was found to be methylated in vivo and the incorporated [3H]methyl groups were alkali sensitive, (iii) this same set of membrane proteins incorporated methyl groups from Sadenosylmethionine in vitro, and (iv) out of a total of eight generally nonchemotactic mutants, two were found to swim only in a forward direction and one of these lacked methyltransferase activity. Analysis of in vivo and in vitro methylation in synchronized cultures showed that the methylation reaction is lost when the flagellated swarmer cell differentiates into a stalked cell. In vivo methylation reappeared coincident with the biogenesis of the flagellum just prior to cell division. In vitro reconstitution experiments with heterologous cell fractions from different cell types showed that swarmer cells contain methyltransferase and their membranes can be methylated. However, newly differentiated stalked cells lack methyltransferase activity and membranes from these cells cannot accept methyl groups. These results demonstrate that MCP methylation is confined to that portion of the cell cycle when flagella are present.
The incorporation of [32P]phosphate by unfertilized mouse ova and 1-cell embryos has been studied. [3ZP]Phosphate was incorporated into material insoluble in cold trichloroacetic acid by unfertilized ova throughout the period 1 -6 h after ovulation with the lowest level of incorporation occurring 2 -4 h after ovulation. Incorporation of label by 1 -cell embryos was highest soon after fertilization but rapidly fell to a minimum within 2 h before rising to a second maximum at about the time of commencement of pronuclear DNA synthesis. A variety of extraction procedures failed to liberate labeled RNA from ova or embryo lysates and less than 10% of the incorporated label was soluble in chloroform/methanol. The 32P-labeled material precipitated from embryo lysates by ethanol/sodium acetate, pH 5 was resistant to snake venom phosphodiesterase before or after digestion with pronase. However, 32P-labeled material insoluble in cold trichloroacetic acid was hydrolysed by hot alkali to 32Pi and by 6 M HC1 to 32P-labeled serine and threonine phosphates and 32Pi thus showing that [32P]phosphate was incorporated solely into protein. Paper electrophoresis and thin-layer chromatography showed that 32P-labeled diphosphates and triphosphates but not monophosphates were present in the 32P-labeled soluble fraction from embryo lysates. 32P-labeled nucleoside monophosphates were also absent from the snake venom phosphodiesterase digest of the labeled soluble fraction but 32Pi and 32PP, were products of hydrolysis. Those results show that the mouse ovum does not contain a store of nucleosides, that pathways for mononucleotide synthesis are not functional in the I-cell embryo, and suggest that the ovum contains a large endogenous store of nucleotides.In most mammals ovulation occurs after the oocyte has reached the metaphase stage of the second meiotic division. Fertilization of the ovum normally takes place within a few hours after ovulation with the entry of the sperm initiating a series of functional and structural changes in the ovum. These include the induction of the block to polyspermy, the completion of the second meiotic division and extrusion of the second polar body, formation of pronuclei and pronuclear DNA synthesis, syngamy and the first cleavage of the embryo. The ovum begins to degenerate several hours after ovulation [I], the period during which the ovum can be fertilized being short. Any delay in fertilization of ova results in a decrease in their fertilizability, and fertilization which occurs towards the end of the fertile period is associated with an increased incidence of chromosomal anomalies, abnormal fetal development and spontaneous abortions [2 -71.Enzymes. Snake venom phosphodiesterase (EC 3.1.4.1); ribonuclease A (EC 3.1.4.22).The difficulty in obtaining large numbers of mammalian ova and embryos has hampered biochemical studies and relatively little is known about metabolic events in unfertilized ova and 1-cell embryos. Most biochemical studies of mammalian ova and 1-cell embryos have centered on those of ...
Unfertilized mouse ova incorporated [3H]adenosine into cold trichloroacetic acid insoluble material at a level which was highest 3-5 h after ovulation but which decreased sharply thereafter. Not all of the [3H]adenosine-labeled material was released into the aqueous layer by chloroformphenol extraction of labeled ova lysates. The labeled material in the aqueous layer was hydrolyzed by ribonucleases A and TI to [ 3 H ] a d e n~~i n e and a fragment which was converted by 0.3 M K O H to 3'-[3H]AMP and [ 3 H ] a d e n~~i n e in the ratio of 12.5/ 1. [3H]Adenosine-labeled material associated with protein was isolated by virtue of its resistance to ribonucleases T2 and U2. This material was released into the supernatant following incubation of the protein fraction with hydroxylamine, alkaline buffers, or 0.3 M K O H and was identified as oligo(ADP-ribose) with average chain length of 4-5 units by B i o c h e m i c a l studies of fertilization and embryonic development have concentrated on the echinoderm and amphibianrather than mammals because of the difficulty in collecting large numbers of mammalian ova and embryos in a short time, and the lack of suitable media and methods for their in vitro culture. The technique for superovulation (Gates, 1971) has enabled the collection of larger numbers of ova and embryos, and the development of culture methods using chemically defined media which support embryonic development through
The incorporation of [3H]adenosine into cold trichloroacetic acid (TCA) insoluble material by the mouse 1-cell embryo has been studied. Incorporation of label was high immediately after fertilization, then decreased over the next 7 h with the sharpest decline occurring 3—5 h after fertilization. A small maximum was observed at the time of pronuclear DNA synthesis. Actinomycin D at a concentration which inhibited the cleavage of 1-cell embryos by 50 % had little effect on this incorporation, which in the period 1—6 h post-fertilization was shown by autoradiography to be confined to the ooplasm of the newly fertilized ovum. [3H]Adenosine and poly ([H]A) were released from embryo RNA labelled 1—3 h after fertilization with [3H]adenosine by digestion with a mixture of ribonucleases A and T1. The poly ([3H]A) segments were hydrolysed by alkali to 3′-[3H]AMP and [3H]adenosine ([3H]AMP/[3H]adenosine = 5/1), and by snake venom phosphodiesterase to 5′-[3H]AMP but very little [3H]adenosine. These results suggest that adenylation of RNA occurs soon after fertilization, that this is a cytoplasmic event, and that most of the newly synthesized poly ([3H]A) segments are joined to pre-existing poly (A) tracts. The unusual polynucleotide, poly (ADP-ribose), identified by its resistance to alkali and the release of 2′-(5″-phosphoribosyl)-5′[3H]AMP on incubation with snake venom phosphodiesterase, was also found in the ribonuclease digest.
The activity of the embryonic genome prior to the first cleavage has been assessed by studying the uptake of [3H]uridine, its phosphorylation and incorporation into RNA by mouse one-cell embryos. One-cell embryos incorporated [3H]uridine linearly into cold trichloracetic acid (TCA) insoluble material at a low level 1–9 h post fertilization. The incorporation of [3H]guanosine was also low but followed a biphasic curve which had a steeper slope at 1–3 h than during the period 4–9 h post fertilization. Unfertilized mouse ova incorporated very little [3H]uridine or [3H]guanosine into TCA insoluble material, and much of this was RNase insensitive. Dimethyl sulfoxide (DMSO) enhanced the uptake of [3H]thymidine and its incorporation into pronuclear DNA by one-cell embryos, but had no effect on the incorporation of [3H]uridine by them, or of [3H] uridine and [3H]guanosine by unfertilized ova. The uptake and incorporation of [3H] guanosine by one-cell embryos were enhanced by DSMO, but only during the period 1–3 h post fertilization. Sugar derivatives of UDP, and UMP, UDP, UTP, CMP, CDP and CTP have been identified in the soluble fraction obtained from mouse one-cell embryos incubated with [3H] uridine 1–3 h post fertilization. Very little of the [3H] uridine taken up by the embryos is present as [3H] UTP, or [3H] CTP; most is found as [3H] UMP or [3H] UDP or as the sugar derivatives. Alkaline or ribonuclease (A, T1 and T2) hydrolysis of the 3H-labeled ethanol insoluble material precipitated from the lysate of one-cell embryos incubated with [3H] uridine 1–3 h post fertilization liberated radioactive cytidine and uridine-3'-phosphates. This demonstrates that [3H] uridine is incorporated into an internal position in RNA and suggests that RNA synthesis does occur in the one-cell embryo 1–3 h post fertilization. Since pronuclei of one-cell embryos incubated with [3H] uridine were not labeled it appears, however, that the RNA synthesized at the one-cell stage is not a product of the embryonic genome.
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