The expression of β-glucuronidase during preimplantation development of mouse embryos

β-Glucuronidase activity was measured in mouse embryos during the preimplantation period of development by using a microfluorometric assay. A 100-fold increase in activity was observed between 57 (8-cell stage) and 84 hr (morulae) of development. Activity changes between 30 and 60 hr were also significant. Genetic variants of β-glucuronidase occur between the strains of mice $\text{C57BL}\text{6J}$ and $\text{C3H}\text{HeJ}$ which differ in levels of activity and heat denaturation kinetics. Activity changes and heat denaturation kinetics of β-glucuronidase in $\text{C57BL}\text{6}\text{,}\text{C3H}\text{HeJ}$ and F₁ hybrid embryos were compared, and it was demonstrated that paternal genes were expressed during the 100-fold increase in activity and that embryonic genes may be functioning between 30 and 60 hr of development.     

In vitro development of early postimplantation rat embryos

Rat embryos explanted at $\text{7}\text{1}\text{2}$ or $\text{8}\text{1}\text{2}$post coitum were cultured throughout the major stages of organogenesis in a system of rotating bottles containing heat-inactivated, immediately centrifuged (I.C.) serum. About 80% of the $\text{8}\text{1}\text{2}\text{-}\text{day}$ explants and 50% of the $\text{7}\text{1}\text{2}\text{-}\text{day}$ explants developed a blood circulation in the yolk sac; in these embryos, organogenesis and growth rates were similar to those of embryos in vivo. In cultures continued for 4 or 5 days, many of the embryos developed 30–40 somites. There was little difference in the subsequent development of embryos cultured in maternal serum or male serum during the egg-cylinder stage except for a possible decrease in the frequency of normal axial rotation in embryos from the male serum. Development in rotator bottles was much better than in watchglass cultures.     

Effect of removing the zona pellucida on development of hamster and bovine embryos invitro and invivo

Uterine stage embryos collected from the hamster (8-cell) and cow (morula, early blastocyst) were monitored for development $\text{in}$$\text{vitro}$ (embryo culture) and $\text{in}$$\text{vivo}$ (embryo transfer) following premature removal of the zona pellucida.Removal of the zona pellucida did not significantly affect $\text{in}$$\text{vitro}$ development to the blastocyst stage of (1) 8-cell hamster embryos (zonae removed by a combined enzymic-mechanical procedure), (2) bovine morulae (zonae removed by mechanical means only) (3) early bovine blastocysts (zonae removed by the enzymic-mechanical technique).Zona-free hamster embryos formed significantly fewer viable fetuses than did zona-intact embryos. The lower incidence of fetal development observed following transfer of zona-free 8-cell hamster embryos may have resulted in part from the formation of chimeras by fusion of these embryos $\text{in}$$\text{utero}$. Such fusion was observed to occur $\text{in}$$\text{vitro}$ between zona-free embryos placed in close proximity. The proportion of pregnancies resulting from transfer of bovine blastocysts cultured from zona-free morulae was similar to that of zona-intact embryos.In this study we have demonstrated that (1) enzymic and mechanical procedures used to remove zonae pellucidae from uterine-stage hamster and bovine embryos do not adversely affect subsequent development of these embryos $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ and (2) zonae pellucidae are not required for normal development of these embryos. These findings have implications for microsurgery of mammalian embryos and for embryo transfer.     

Idiopathic paraproteinemia. III. Increased frequency of paraproteinemia in thymectomized aging C57BL/KaLwRij and CBA/BrARij mice

The influence of thymectomy on the appearance of idiopathic paraproteinemia (IP) during aging was investigated in mice of the C57BL/KaLwRij and the CBA/BrARij strains, which under normal conditions develop IP in high and low frequency, respectively. Compared with sham-thymectomized mice, C57BL mice thymectomized at a young adult age showed a markedly increased frequency and an earlier onset of IP during aging; this was even more pronounced in neonatally thymectomized mice. A similar effect of thymectomy was also observed in mice of the CBA strain. Restriction in the heterogeneity of the serum immunoglobulins and the appearance of transient homogeneous Ig components was another frequent finding and this often preceded the appearance of IP in mice of both strains. Thymectomy did not substantially influence either the incidence of paraproteinemias due to a B cell malignancy or the isotype distribution among the paraproteins produced. The results are compatible with the hypothesis that IP develops in three stages as a consequence of an age-related immunnodeficiency that primarily affects the T immune system.     

Effects of the brachyury (T) mutation on mitotic activity in the neural tube.

Mitotic activity in the neural tube was examined in +/+, $\text{T}\text{+}$ and $\text{T}\text{T}$ mouse embryos. The number of mitotic figures per unit area of ependymal layer (area index) was higher in $\text{T}\text{+}$ embryos than in +/+ embryos. In +/+ embryos, area index was higher in the dorsal part of the neural tube than in the ventral part, whereas this pattern is disordered in $\text{T}\text{+}$ embryos. In $\text{T}\text{T}$ embryos, these values fluctuate. The process of nuclear migration accompanying cell division seems to be normal in the neural tube of $\text{T}\text{+}$ and $\text{T}\text{T}$ embryos.     

Trichinella spiralis: Characterization and strain distribution of rapid expulsion in inbred mice

Appropriately immunized mice display a response that is biologically equivalent to rat rapid expulsion. Only two inbred strains ($\text{NFR}\text{N}\text{and}\text{NFS}\text{N}$ derived from NIH Swiss mice) have been shown to respond in this manner. Mice of the $\text{Balb}\text{c}$, CBA, $\text{A}\text{He}$, C₃H, SJL, or C₅₇Bl strains are “nonresponders” which require approximately twice as much intestinal exposure (in days) to Trichinella spiralis to elicit a response half as effective. Genetically, the responder is dominant, autosomal, and does not appear to be linked to the MHC. The characteristics of mouse and rat rapid expulsion of T. spiralis are not identical but share these features: initial rejection within 24 hr of challenge; a rejection efficiency >90%, from 1 to 5 weeks after the primary; induction of response does not require exposure to the complete infection; rapid expulsion is immunologically specific for preadults; adult worms are resistant. While a genetic basis for responsiveness exists in mice there is, as yet, no evidence for genetic control in rats. In both mice and rats, rapid expulsion is distinguished from the intestinal hyperreactivity associated with rejection of the primary infection by the kinetics and amplitude of the rejection of transplanted adult worms.     

Schistosoma mansoni: Splenic lymphocyte responses of mice after initial exposure to highly irradiated cercariae

Inbred $\text{C}\text{57}\text{B}\text{1}\text{6}$ and CBA mice were immunized with ⁶⁰Co-irradiated (50 kR) Schistosoma mansoni cercariae. Based on the percentage reduction from controls in the numbers of adult parasites developing from a challenge cercarial exposure, the level of protection among immunized $\text{C}\text{57}\text{B}\text{1}\text{6}$ mice ranged from 56 to 74%, and among immunized CBA mice from 10 to 27%. In a longitudinal study, parallel in vitro comparisons of mitogen- and antigen-stimulated lymphocyte proliferative responses were performed with spleen cells from immunized and control mice of both strains. In contrast to decreased mitogen reactivity during a chronic, patent infection, immunization with irradiated cercariae resulted in no alteration in PHA and LPS responses in the reactivity of either strain. A vigorous antigen-specific reactivity was noted in the responses of immunized CBA mice. Additionally, a biphasic pattern of responsiveness characterized the CBA responses to antigens of cercarial, adult worm, or schistosomal egg origin. In comparison, there was a greatly diminished reactivity in immunized $\text{C}\text{57}\text{B}\text{1}\text{6}$ mice to the same antigens. Therefore, no obvious correlation existed in this model between the relative magnitude of antigen-specific responses between the two strains and the level of anti-schistosome immunity induced.     

Evidence for dosage compensation of an X-linked gene in the 6-day embryo of the mouse.

The time at which dosage compensation of an X-linked gene in the mouse is established has been estimated by measuring the activity levels of two glycolytic enzymes, phosphoglycerate kinase (EC 2.7.2.3) and triosephosphate isomerase (EC 5.3.1.1), during early development of embryos from $\text{X}\text{X}$ and $\text{X}\text{O}$ mice. During preimplantation development the level of phosphoglycerate kinase in embryos from $\text{X}\text{X}$ mice was constant for the first 48 hr of development (2.55–2.71 nmoles/hr/embryo) and then dropped to one-half the earlier level (1.44 nmoles/hr/embryo) by 72 hr of development. The general developmental profile of phosphoglycerate kinase was similar in embryos from $\text{X}\text{O}$ mice; however, the absolute level of enzyme activity was reduced to approximately 1.4 nmoles/hr/embryo during the first 48 hr of development and to 0.9 nmoles/hr/embryo by 72 hr of development. These differences in phosphoglycerate kinase levels between embryos from $\text{X}\text{X}$ and $\text{X}\text{O}$ mice disappeared between the blastocyst and egg cylinder stages (150 hr postplug) during which time the activity levels had increased to 183 and 193 nmoles/hr/egg cylinder for embryos from $\text{X}\text{O}$ and $\text{X}\text{X}$ mice, respectively.The activity level for triosephosphate isomerase in embryos from $\text{X}\text{X}$ and $\text{X}\text{O}$ mothers did not differ at any stage of development; this suggests that the gene coding for triosephosphate isomerase is autosomal. In addition the level of activity remained constant during preimplantation development (approximately 3 nmoles/hr/embryo) and then, like phosphoglycerate kinase, increased 100-fold between the blastocyst and egg cylinder stages.The frequency distribution of the activity ratio (triosephosphate isomerase to phosphoglycerate kinase) in the egg cylinder of individual embryos from both $\text{X}\text{X}$ and $\text{X}\text{O}$ mothers was determined in order to detect differences among $\text{X}\text{X}\text{,}\text{X}\text{O}$ and $\text{X}\text{Y}$ embryos. These frequency distributions were unimodal, and hence these results coupled with the gene dosage differences observed during preimplantation development indicate that dosage compensation for an X-linked gene has been established in the 6-day mouse embryo.     

Genetic expression of aryl hydrocarbon hydroxylase activity in the mouse: Distinction between the “responsive” homozygote and heterozygote at the Ah locus

β-Napththoflavone administration induces certain monooxygenase activities, such as aryl hydrocarbon (benzo[a]pyrene) hydroxylase, and cytochrome P₁-450 formation in the “responsive” C57BL/6 and C3H/He inbred mouse strains, whereas these changes are absent or relatively small in the so-called nonresponsive $\text{DBA}\text{2}$ inbred strain. Dose-response curves—with the use of large numbers of animals of the same age and sex and with either β-naphthoflavone or the much more potent 2,3,7,8-tetrachlorodibenzo-p-dioxin as inducer—reveal a small, but statistically significant, difference in the hydroxylase induction between the $\text{C}\text{57}\text{BL}\text{6}\text{J}$ homozygote and the ($\text{C}\text{57}\text{BL}\text{6}\text{J}$)($\text{DBA}\text{2}\text{J}$)F₁ heterozygote in liver, kidney, bowel, and lung. The (C3H/HeJ)($\text{DBA}\text{2}\text{J}$)F₁ heterozygote displays additive inheritance in each of these same tissues.     

Development of teratomas from yolk sac of genetically sterile embryos

Yolk sac-derived teratomas are composed of various well-differentiated tissues. These tissues must be derived from multipotent cells. To exclude a germ cell origin for these teratomas we used $\text{Steel}\text{-}\text{Sl}\text{+}$ females copulated with $\text{Sl}\text{+}$ males. The embryos generated from such mating comprise 25% $\text{Sl}\text{Sl}$ sterile embryos, deficient in primordial germ cells, 25% normal embryos (+/+), and 50% heterozygotes ($\text{Sl}\text{+}$). The results indicate that the genotype of the embryos does not influence the development of teratomas. Displaced yolks sacs belonging to genetically sterile embryos developed into teratomas as frequently as those from heterozygotes and from genetically normal embryos.     

Autonomy for a specific gene product in oocytes: Experimental evidence in the polychaetous annelid,Platynereis dumerilii

Oocytes of Platynereis dumerilii in early vitellogenesis were injected into female worms with oocytes of similar diameter. The donor oocytes were labeled by the or gene controlling eye pigmentation and, after some weeks of growth, were spawned together with the host oocytes. In most cases, a few donor progeny could be found among the offspring produced by the hosts. Donor progeny were examined with respect to an or gene-dependent maternal effect which normally causes wild-type eye color in homozygous ($\text{or}\text{or}$) larvae originating from the crossings of heterozygous ($\text{or}{}^{\mathrm{+}}\text{or}$) females and homozygous ($\text{or}\text{or}$) males. This maternal effect was absent from homozygous ($\text{or}\text{or}$) larvae derived from homozygous ($\text{or}\text{or}$) donor oocytes which had developed in heterozygous females. Conversely, this maternal effect was observed in homozygous ($\text{or}\text{or}$) larvae derived from heterozygous ($\text{or}{}^{\mathrm{+}}\text{or}$) donor oocytes which had developed in homozygous ($\text{or}\text{or}$) host females. It is concluded that the oocyte genome is active at the or⁺ locus during oogenesis and that the oocyte is autonomous with respect to the product of synthesis of the or⁺ locus. In the present case, the “maternal effect” is therefore caused by synthetic activity in the growing oocyte. The results are discussed with respect to current information on gene products from animal genomes.     

Effects of the Brachyury (T) mutation on morphogenetic movement in the mouse embryo

$\text{T}\text{T}$ embryos have been first distinguishable at 8 days post coitum by their gross morphological abnormalities. By quantitative morphometry of histological sections, anomalies in the homozygotes were expressed numerically. At 8 days p.c., morphologically identifiable T-homozygotes had an increased number of ectodermal and a reduced number of mesodermal cells compared to the wild type. At 7 days p.c., embryos with a low mesoderm/ectoderm ratio were found only in litters of $\text{T}\text{+}\text{×}\text{T}\text{+}$ matings at the expected frequency. At 6 days p.c., one-fourth of the embryos in $\text{T}\text{+}\text{×}\text{T}\text{+}$ litters showed a delay in the transition from cuboidal to squamous endoderm. No such embryos were found in the +/+ × +/+ matings. In 6-, 7-, and 8-day mutant embryos, cells proliferated at statistically normal rates. Therefore, it may be said that advanced morphological irregularities of 8-day homozygotes cannot be accounted for by anomalies in cell proliferation. When the total cell number was 5 × 10⁴/embryo (8 days), a sudden change was observed in the regional distribution of mesodermal and ectodermal cells along the anteroposterior axis of $\text{T}\text{T}$ embryos. Since no regional difference in the cell cycle time was observed, these abnormalities may best be explained by anomalies in cell migration. These results strongly suggest abnormal morphology of $\text{T}\text{T}$ mutants resulting from defects in morphogenetic movement.     

The secondary attachment site for bacteriophage lambda in the proA/B gene of Escherichia coli

We have determined the nucleotide sequence of a secondary λ attachment site in $\text{pro}\text{A}\text{B}$, a site that accounts for 3% of lysogens isolated from Escherichia coli strains deleted for the primary site. Direct sequence analysis of the transducing bacteriophages carrying the left and right att junctions, as well as the recombinant pro⁺ phage reveals that the $\text{pro}\text{A}\text{B}$ site shares an 11-nucleotide interrupted homology with the core sequence of the primary site. We have compared the $\text{pro}\text{A}\text{B}$att site with other secondary attachment sites to gain insights into the structural features important for λ integration.     

Kinetics of bidirectional active sodium fluxes in the toad bladder

The kinetics of isotopic Na⁺ flows was studied in urinary bladders of toads from the Dominican Republic. Initial studies of the potential dependence of passive serosal to mucosal ²²Na⁺ efflux demonstrated the absence of isotope interaction and/or other coupling with passive Na⁺ flow. The electrical current $\text{I}$ and mucosal to serosal ²²Na⁺ influx were then measured with transmembrane potential clamped at $\text{Δψ = 0, 25, 50, 75 or 100}\text{mV}$. Subsequent elimination of active Na⁺ transport mucosal amiloride permitted calculation of the rates of active Na⁺ transport $\text{J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>a</mtext>}}$ and active and passive influx and $\text{J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>a</mtext>}}$ and $\text{J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>p</mtext>}}$. The results indicate that for Dominican toad bladders mounted in chambers only Na⁺ contributes significantly to transepithelial active ion transport; hence $\text{J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>a</mtext>}}\text{= J}{}^{\mathrm{<mtext>a</mtext>}}$. $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ was abolished at $\text{Δψ = E = 96.3 ± 1.9}\text{(S.E.) mV}$. As Δψ approached $\text{E}$, active efflux $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ became demonstrable. At $\text{Δ = 100}\text{mV}\text{,}\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ exceeded $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$, so that $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ was negative. Experimental values of $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ agreed well with theoretical values predicted by a thermodynamic formulation: $\text{J}{}_{\mathrm{<mtext>exp</mtext>}}{}^{\mathrm{<mtext>a</mtext>}}\text{= 0.985}\text{J}{}_{\mathrm{<mtext>theor</mtext>}}{}^{\mathrm{<mtext>a</mtext>}}\text{(r = 0.993)}$. The dependence of $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ on Δψ is curvilinear.     

Immune responses to hapten--lipopolysaccharide conjugates. 1. Modulation of in vivo antibody responses to the polysaccharide

The immune responses of mice to various lipopolysaccharides (LPS) and hapten-LPS conjugates were compared. We found that some strains of mice ($\text{A}\text{J}$ and BALB/c) produced equivalent amounts of anti-LPS antibody after the injection of either LPS or hapten-LPS conjugates. In contrast, however, other strains of mice (C57BL/6J, C3H/St, DBA/1J, DBA/2J, and Swiss) produced fewer anti-LPS-antibody-secreting cells after stimulation with hapten-LPS conjugates than did mice injected with unsubstituted LPS. The covalent coupling of hapten to LPS changed neither the mitogenic capacity nor the antigenicity of the LPS. The differences in the magnitude of antibody responses to hapten-LPS and LPS in these latter strains of mice occurred in the absence of mature T lymphocytes and was restricted to the primary immune response. Furthermore, these differential responder mice (C57BL/6J) did produce anti-LPS antibody when primed with LPS before challenge with the hapten-LPS conjugate. These data are discussed with respect to both the modulatory capacity of the hapten-LPS in the regulation of the primary immune response to LPS and the biochemical and structural requirements of the hapten-LPS conjugate for immunogenicity.     

Interactions of cadmium and zinc in cultured rat embryos

Rat embryos were cultured in serum taken from animals dosed with cadmium, or serum with cadmium added $\text{in}$$\text{vitro}$ in the presence or absence of additional zinc. Embryos explanted at day ten and grown in serum taken from animals sooner than 4 h after dosing had a reduced DNA content after 24 h culture. In one-hour serum, the yolk sac had become thick and brittle. Zinc ameliorated the effects but had no stimulatory effect on post eight-hour serum when serum zinc levels were at their lowest. The hypothesis that cadmium induces a maternal zinc deficiency sufficient to cause teratogenic changes could not be sustained. Embryos explanted at nine days were much more susceptible to cadmium added $\text{in}$$\text{vitro}$ than ten-day embryos. The principal anomaly, apart from a reduced DNA content, was a thickening of the yolk sac similar to that seen in embryos grown in serum taken from animals one hour after cadmium dosing. Addition of zinc to the medium prevented both of these effects. The suggestion is made that the cadmium-induced dysgenesis of the yolk sac precludes appropriate embryonic nutrition.     

Embryonic development in repeat breeder and virgin heifers seven days after insemination

The aim of this study was to compare the development of embryos from repeat breeder heifers with that of embryos from virgin heifers at 7 days after standing heat. A total of 23 repeat breeder heifers (RBH) and 18 virgin heifers (VH) were utilized. The heifers were between 16 and 30 months of age and most of them were of the Swedish Red and White Breed. Two RBH were heterozygous for the $\text{1}\text{29}$ chromosome translocation, one RBH was a trisomy X and all the other heifers had normal karyotypes. All heifers were inseminated with frozen semen from the same bull and all inseminations were performed by the author. The fertility of the bull was above the average for the AI association to which it belonged. Embryos were collected by a non-surgical technique (89) or after slaughter (19). The morphology of the embryos was examined under a phase-contrast microscope and they were classified as being normal (N), morphologically deviating (MD) or degenerated (D). Thirteen embryos from RBH and 15 from VH were examined for total cell numbers after examination of their morphology.There was no significant difference in recovery rates of embryos between RBH (68%) and VH (76%) but independent of collection method the recovery rate of embryos from VH was numerically higher. The fertilization rate was high in both RBH (89%) and VH (97%). Seventyfour percent of the embryos collected from VH were normal ($\text{23}\text{31}$) while only 28% ($\text{11}\text{40}$) of the embryos collected from RBH had a normal morphology. The difference in number of normal embryos recovered from the two groups of heifers was highly significant (P < 0.005). Exclusion of the RBH heifers with deviating karyotype did not influence this difference. However, there was a tendency to a higher incidence of fertilization failure and morphologically deviating embryos in these heifers. The N embryos had significantly higher total cell numbers (P < 0.005) than the MD embryos but there was no significant difference in total cell numbers between N embryos from RBH or VH.The results of this study strongly indicate a higher incidence of abnormal embryos in RBH than in VH. It is likely that these deviations are followed by an increased incidence of early embryonic death.     

Ontogeny of the retina and optic nerve of Xenopus laevis: IV. Ultrastructural evidence of early ganglion cell differentiation

Ultrastructural evidence indicates that Xenopus retinal ganglion cell axons differentiate early, between stages 28 and 32. Light microscope studies indicated the presence of argryophilic material in the ventral retina and optic stalk of early embryos. Ultrastructural analysis of this region confirmed the presence of axons in the stalk and interstices of ventral retinal cells. Axons containing aligned microtubules and neurofilaments and elongated mitochondria with a paucity of other cell inclusions are found with increasing frequency in the ventral retina from stages 28 through $\text{33}\text{34}$. Central and dorsal regions of the retinas examined show little or no evidence of axons. A discrete, small bundle of axons is found in the optic stalk of stage 28 embryos and by stage $\text{30}\text{31}$ the number of axons in bundles has increased, suggesting early fasciculation. Between stages 28 and $\text{33}\text{34}$ (± 12 hr) extracellular space surrounding early axons diminishes and processes from neuroretinal cells in contact with axons surround developing axon bundles. The evidence presented suggests that axon initiation occurs in stages much earlier than previously reported. Other investigators have failed to detect ganglion cell differentiation prior to stage 32 possibly because they examined regions of the retina with few axons. Thus, experiments which rotate the retina in the orbit may have to be reevaluated since regenerating axons may use previously established pathways to organize and “home in” on tectal target cells.     

Localization at the ultrastructural level of maternality derived enzyme and determination of the time of paternal gene expression for acid phosphatase-1 in Drosophila melanogaster.

Ultrastructural histochemistry instead of acrylamide gel electrophoresis (see R. Yasbin, J. Sawicki, and R. J. MacIntyre, 1978, Develop. Biol. 63, 00-00) is used to determine the time of paternal gene expression for the enzyme acid phosphatase-1 of Drosophila melanogaster in $\text{Acph-1}{}^{\mathrm{n}}\text{Acph-1}{}^{\mathrm{B}}$ embryos in which the null allele is derived from the female parent. Timed embryos were histochemically stained for acid phosphatase activity according to the lead phosphate method of Gomori and were examined at the ultrastructural level. Enzyme activity, resulting from activation of the paternal Acph-1 gene, is detected as early as 5 hr after fertilization. Maternally derived enzyme in $\text{Acph-1}{}^{\mathrm{B}}\text{Acph-1}{}^{\mathrm{B}}$ embryos is found principally in the yolk regions and around invaginations. This suggests that acid phosphatase-1 functions in yolk digestion and in cell movements during early embryogenesis.     

Capsule degradation in azotobacter

Enzymic degradation of $\text{Azotobacter}$ capsular polysaccharide by the depolymerases from azotophage lysates of $\text{A}$. $\text{vinelandii}$, and from a strain of $\text{A}$. $\text{chroococcum}$ was examined. The molecular size of the capsular polysaccharide was examined by molecular sieve chromatography both before and after exposure to the capsule depolymerase. The depolymerase appears to attack the polysaccharide substrate in a random fashion which results in polysaccharide fragments of a random size distribution. The ester linkages and hexuronic acids were proportionally the same in all fractions before degradation, but ester linkages were absent from the smaller polysaccharide molecules after enzyme action. The ester linkages were not the substrate bonds broken by the enzyme, but, in the case of some azotophage enzymes, they appeared to play a role in enzyme activity, possibly as recognition sites.