Developmental changes in chromatin organization in rat cerebral hemisphere neurons and analysis of DNA reassociation kinetics

Previous reports have demonstrated that neuronal nuclei of rabbit, mouse and rat cerebral hemispheres exhibit a short DNA repeat length of 160 bp compared to the more typical repeat size of 200 bp found in glial nuclei and other cell types of higher eukaryotes. In this study we report that the conversion of chromatin to a short DNA repeat length in rat cerebral hemisphere neurons is a gradual process which begins between the first and second day after birth and is complete by 8 days. In these neurons, histone H1 appears to be less accessible to degradation by trypsin in the newborn rat brain compared to the 8 day old rat. This suggests that the developmental shift to a short DNA repeat length may be accompanied by a dispersal or decondensation of neuronal chromatin which results in an increased accessibility of neuronal histone H1 to degradation by trypsin. The increase in nuclear DNA content to 3.5C which has been reported in rat cortical neurons during early postnatal development does not appear to be associated with a selective amplification of a subset of DNA sequences as determined by DNA reassociation kinetics.     

Prenatal appearance of a short nucleosomal DNA repeat length in neurons of the guinea pig cerebral cortex

The organization of chromatin in neurons of the cerebral cortex of the guinea pig brain was analyzed by digesting isolated nuclei with micrococcal nuclease. During development, cortical neurons were observed to undergo an alteration in chromatin structure which results in an atypically short nucleosomal DNA repeat length of 164 bp. This change in chromatin organization occurs postnatally in certain mammals but in the guinea pig it takes place prior to birth between days 32 and 44 of fetal development. This suggests that the appearance of the short nucleosomal DNA repeat length in cortical neurons correlates to a particular stage of differentiation of cortical neurons rather than to the event of birth.     

The repetitive sequence structure of component alpha DNA and its relationship to the nucleosomes of the African green monkey.

An analysis of the repeat structure of the highly repetitive sequence, component α DNA of the African green monkey, shows that the DNA contains restriction sites for EcoRI, $\text{Eco}\text{RI}{}^1$, HindIII and HaeIII. All four restriction enzyme activities indicate a basic repeat length of 176 ± 4 base-pairs. In addition to primary $\text{Eco}\text{RI}{}^1$ and HindIII sites, about 59% of the repeat sequences contain secondary $\text{Eco}\text{RI}{}^1$ sites and about 36% of the repeat sequences contain secondary HindIII sites. The secondary sites are located less than 176 base-pairs from the primary sites and their cleavage yields several complex series of minor, intermediate segments in gels of the partial $\text{Eco}\text{RI}{}^1$ or HindIII digests. Cleavage at the secondary sites yields segments shorter than the unit monomer in the limit digests. The sites for EcoRI, $\text{Eco}\text{RI}{}^1$, HindIII and HaeIII have been mapped within the repeat unit.Treatment of the monkey nuclei with micrococcal nuclease at 2 °C and in the presence of 80 mm-NaCl reveals two distinct populations of nucleosomes. One population contains bulk DNA sequences, and after cleavage with micrococcal nuclease this population yields heterogeneous segments of DNA spanning 180 to 200 base-pairs in length. The other population contains component α sequences and after cleavage with micrococcal nuclease yields homogeneous segments of component α DNA that are exact multiples of the basic sequence repeat unit of 176 base-pairs. Thus, the cleavage by micrococcal nuclease of nucleosomal arrays containing component α sequences is as regular and precise as the cleavage of the purified DNA by the restriction enzymes. The resolution of the two distinct subsets of nucleosomes in the monkey nuclei is dependent upon the conditions of ionic strength and temperature employed during the nuclear isolation and the micrococcal nuclease digestion.These observations are consistent with a phase relation between the component α repeat sequences and the associated nucleosomal proteins (Musich et al., 1977b). They are also in accord with the hypothesis that the subunit structure of constitutive heterochromatin modulates or determines the repeat sequence structure and hence, the evolution of many highly repetitive mammalian DNAs (Maio et al., 1977).     

A spectrofluorimetric investigation of calf thymus DNA modified by BP diolepoxide and 1-pyrenyloxirane

7β,8α-Dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP diolepoxide, $\text{1}$) and 1-pyrenyloxirane ($\text{2}$) bind chemically to calf thymus DNA. The fluorescence efficiency of pyrenyl groups in mutagen modified DNA varies appreciably with its conformation and decreases in the order: pyrenees, modified denatured DNA and modified native DNA. A particularly interesting observation is that the fluorescence efficiency of mutagen modified DNA intensifies substantially upon denaturation. Our results suggest that the pyrenyl groups in mutagen modified DNA are intercalated between the base pairs of DNA. Since both $\text{1}$ and $\text{2}$ are powerful frame-shifting mutagens for S. typhimurium TA-98, the intercalative covalent binding of these compounds to DNA may provide a molecular basis for their mutagenic activity.     

Effects of 19-hydroxy-prostaglandins on oviductal and uterine motility

The effects of 19-hydroxy-prostaglandins (19-OH-PGs) were tested $\text{in}$$\text{vivo}$ on the rabbit oviduct and uterus and on the rhesus monkey ($\text{Macaca}$$\text{mulatta}$) uterus. The 19-OH-PGEs suppressed spontaneous oviductal and uterine activity in the rabbit. The qualitative effect on the rabbit oviduct of 19-OH-PGEs was similar to that of PGE₂. However, the typical response of the rabbit uterus to PGE₂ was an increase in muscle activity. With regard to the rabbit oviduct, 19(R)-OH-PGE₂ was as potent as PGE₂, but 19(S)-OH-PGE₂ was approximately $\text{1}\text{2}$ as potent as PGE₂. Based on the dose of 19-OH-PGEs usually required to cause a minimal suppression and the dose of PGE₂ required to cause a minimal stimulation of rabbit uterine activity, 19(R)-OH-PGE₂ was twice as potent as PGE₂ while 19(S)-OH-PGE₂ was $\text{1}\text{2}$ as potent as PGE₂. Stimulatory effects on the rabbit oviduct and uterus were observed following administration of 19-OH-PGEs and PGF_2α. The potency on the rabbit oviduct of 19(S)-OH-PGF_2α was about $\text{1}\text{5}$ to $\text{1}\text{10}$ that of PGF_2α; the potency of 19(R)-OH-PGF_2α was about $\text{1}\text{10}$ to $\text{1}\text{20}$ that of PGF_2α. Both 19-OH-PGFs were approximately $\text{1}\text{5}$ to $\text{1}\text{10}$ as potent as PGF_2α on the rabbit uterus. At the doses tested 19-OH-PGFs were inactive on the monkey uterus. Thus, these compounds are at least $\text{1}\text{5}$ as active as PGF_2α. In contrast, 19(R)-OH-PGE₂ had approximately the same potency as PGE₂ in stimulating monkey uterine activity; but 19(S)-OH-PGE₂ was approximately $\text{1}\text{3}$ as potent as PGE₂.     

Chromatin organization in the rat hypothalamus during early development.

The organization of chromatin in neuronal and glial nuclei isolated from different brain regions of rats during development was studied by digestion of nuclei with micrococcal nuclease. A short chromatin repeat length (approx. 176 base-pairs compared with that of glial nuclei from foetal cerebral cortex (approx. 200 base-pairs) was present in hypothalamic neurons throughout the ages studied, which was similar to the repeat length of cortical neurons from 7- and 25-day-old animals (approx. 174 base-pairs). Whereas in cortical neurons the chromatin repeat length shortened from approx. 200 base-pairs in the foetus to approx. 174 base-pairs in the first postnatal week, the short chromatin repeat length of hypothalamic neurons was already present 2 days before birth, indicating that hypothalamic neurons differentiate earlier than cortical neurons during brain development.     

Subunit organization of Euglena chromatin

Digestion of $\text{Euglena}$ nuclei or extracted chromatin with micrococcal nuclease results in the identification of a repeating structure. The DNA repeat size, analyzed on agarose and polyacrylamide gels, is found to be 225±13 base pairs. DNase I digestion produces a serie of fragments multiples of roughly 10 bases. Eventhough pressure shearing is necessary to disrupt the though pellicule of the phytoflagellate, we confirm that, in $\text{Euglena}$, chromatin organization is similar to that of other eukaryotes.     

The epoxide hydrolase catalyzed hydrolysis of trans-3-bromo-1,2-epoxycyclohexane. A direct proof for a general base catalyzed mechanism of the enzymatic hydration

The hydrolysis of (±)-$\text{trans}$-3-bromo-1,2-epoxycyclohexane in the presence of rabbit liver microsomes was investigated, and found to yield, beside $\text{c}$-3-bromocyclohexane-$\text{r}$-1,$\text{t}$-2-diol, 2,3-epoxycyclohexanol. It was demonstrated that the latter compound was the only product of the enzymatic reaction, whereas the diol resulted from a non enzymatic hydration in the reaction medium. These data provide the first direct proof for a general base catalysis in the enzymatic epoxide hydration, previously hypothesized on the basis of several lines of indirect evidence, and disprove alternative mechanisms involving protonation of the oxirane oxygen.     

Molecular structure of 2,2′-anhydro-1-β-D-arabinofuranosyl cytosine hydrochloride (cyclo ARA-C): A highly rigid nucleoside

The molecular structure of cyclo ara-C hydrochloride has been determined by x-ray diffraction methods. The ether linkage between the base and sugar moieties severely restricts the conformation about the glycosyl bond and the mode of sugar puckering. The glycosyl torsion angle (X_CN =299°) lies in a region outside the $\text{anti}\text{and}\text{syn}$ ranges found for the β-nucleosides. The arabinose ring exhibits C(4′)-endo (⁴E) mode of puckering, with a pseudorotation phase angle P of 233°. The positive charge on the base apparently stabilizes the $\text{gauche}\text{-}\text{gauche}$ conformation of the C(5′)-O(5′) bond despite the short contacts between O(5′) and C(2) and N(1) of the base.     

Expression in Escherichia coli of urokinase antigenic determinants

Hybrid genes were constructed between the β-lactamase gene of plasmid pBR322 and enzymatically synthesized DNA sequences coding for urokinase-like material. The recombinant plasmids, pULB1013 and pULB1010, contain DNA inserts of 220 and 535 base pairs respectively, which appear to be in the correct reading frame. The hybrid genes are expressed in $\text{Escherichia coli}$ and their products are specifically immunoprecipitated with antiserum to urokinase. In addition injection into a rabbit of a crude extract from the clone pULB1010 leads to the production of specific antibodies against urokinase.     

The dextro and levorotatory isomers of N-phenylisopropyladenosine: stereospecific effects on cyclic AMP-formation and evoked synaptic responses in brain slices.

The stereoisomers of N⁶-phenylisopropyladenosine elicit accumulations of cyclic AMP in brain slices via interaction with adenosine-receptors. The response in guinea pig cerebral cortical slices and in rat hippocampal slices is blocked by theophylline and potentiated by biogenic amines. A chelator, EGTA, potentiates the response to phenylisopropyladenosine in guinea pig cerebral cortical slices. The $\text{1}$-isomer (EC₅₀ 25 μM) is four- to five-fold more potent than the $\text{d}$-isomer in eliciting accumulations of cyclic AMP in brain slices. In a rat coronal hippocampal slice $\text{in vitro}$, $\text{1}$-phenylisopropyladenosine (IC₅₀ ～ 0.7 μM) reduces the amplitude of evoked synaptic responses generated $\text{via}$ a monosynaptic pathway to the CA1 pyramidal neurons. The $\text{d}$-isomer is nearly one hundred-fold less potent. Thus, the adenosine-receptors involved in the electrophysical response appear much more stereoselective for the $\text{1}$-isomer of phenylisopropyladenosine than the adenosine-receptors involved in cyclic AMP-generation in brain slices.     

Human serum albumin (HSA) decreases prostaglandin A1 (PGA1) metabolism.

PGA₁ was incubated with rabbit renal cortical homogenates containing HSA (0–4.5%). The ability of this tissue to readily metabolize PGA₁ progressively decreased with increasing HSA levels in the incubates The rate of disappearance of ³H-PGA₁ was twice as rapid in rats treated with salicylic acid (S. A.) in comparison to control animals; since only 30% of the injected radioactivity was bound to the plasma of the S.A. treated rats, as compared to 90% bound to control plasma, an association may exist between the degree of binding of ³H-PGA₁ and its rate of clearance. The studies indicate that PGA₁ interaction with HSA decreases its metabolism $\text{in vitro}$, and slows down its clearance $\text{in vivo}$, implicating HSA as a possible factor in prostaglandins metabolism $\text{in vivo}$.     

Periodic fluctuations in FSH concentrations during the ovine estrous cycle

Heterologous radioimmunoassays for ovine LH and ovine FSH were validated and used to examine the concentrations of gonadotropins during the estrous cycle. Concentrations of LH were maximal on the day of estrus as previously reported. Concentrations of FSH were minimal 1 or 2 days before estrus, increased markedly during estrus, and fluctuated widely during diestrus. Most ewes ($\text{11}\text{13}$) had periodic waves of FSH occurring at short intervals (3.5–6 days, $\text{3}\text{13}\text{ewes}$), long intervals (10–18 days, $\text{3}\text{13}\text{ewes}$), or at both long and short intervals ($\text{5}\text{13}\text{ewes}$).     

Fluorescence properties of a benzo(a)pyrene 7,8 dihydrodiol 9,10-oxide-DNA adduct. Conformation and effects of intermolecular DNA interactions

The pyrene-like fluorescence of the covalent benzo(a)pyrene diol-epoxide-DNA complex prepared by reacting 7,8,-dihydrodiol 9,10-epoxy benzo(a)pyrene (BPDE) with DNA in aqueous solution $\text{in vitro}$, has been investigated. It is shown that this fluorescence is $\text{sensitive}$ to molecular oxygen, to the concentration of $\text{native}$ DNA and to the ionic strength (KCl concentration), but is $\text{insensitive}$ to the concentration of $\text{denatured}$ DNA. These effects are related to the conformation of the pyrene-like chromophore of BPDE. Most of the fluorescence of a $\text{dilute}$ solution of the DNA-bound benzo(a)pyrene derivative originates from binding sites in which the pyrene moiety is not intercalated between the DNA base pairs, but is located on the outside of the DNA double helix.     

An organ culture system for study of fetal lung development

Fetal rat lungs placed in $\text{in}$$\text{vitro}$ organ culture at 15.5 days gestation grow significantly based on accumulation of DNA and protein. In the experimental system described, DNA accumulated rapidly during the first three days in culture and increased from 4.8 to 15.6 micrograms per lung culture. Protein content increased more slowly and reached a value more than double the initial value after six days in the culture system. Glycogen accumulated in the tissue during the first six days in culture and was depleted during the subsequent culture period, a pattern strikingly similar to that observed during lung development $\text{in}$$\text{vivo}$. Phospholipid accumulation was biphasic with respect to time with an inflection point at about the sixth day of culture. The phosphatidylcholine species synthesized in the culture system $\text{in}$$\text{vitro}$ were similar to those produced $\text{in}$$\text{vivo}$ in fetal lung at 21 days gestation.     

Isolation of a replication-complex from eukaryotes

Treatment of the eukaryotic organism $\text{Tetrahymena pyriformis}$ with low concentrations of Ethidium Bromide causes accumulation of a protein-nucleic acid complex consisting of a DNA polymerase, a RNA polymerase, a deoxyribo-nuclease and a RNA linked DNA fragment. The length of the RNA is about 30 nucleotides, while the DNA part is around 200 nucleotides long. Degradations with ribonucleases and deoxyribonucleases strongly indicate that the RNA exists in a non-hybrid structure with a homogenous base composition and that the DNA is single-stranded. The complex is purified 1100 fold from whole cells and sodium dodecyl sulphate acrylamide gel electrophoresis gives 9 defined bands. The polynucleotide in the isolated complex accounts for only 10^?4 of the total cellular DNA.As the complex contains some of the enzymes essential for discontinous DNA replication, in addition to a RNA linked Okazaki fragment, it is concluded that a highly purified $\text{replication complex}$ has been isolated.     

Stereospecific activity of 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine and comparison of analogs in the degranulation of platelets and neutrophils

1-O-Hexadecyl-2-O-acetyl-$\text{sn}$-glycero-3-phosphocholine (platelet activating factor) stimulated the degranulation of rabbit platelets and human neutrophils, whereas the enantiomer, 3-O-hexadecyl-2-O-acetyl-$\text{sn}$-glycero-1-phosphocholine, was inactive. The analogs compared had the following relative potencies in degranulating platelets and neutrophils: 1-O-hexadecyl-2-O-acetyl-$\text{sn}$-glycero-3-phosphocholine > 1-O-hexadecyl-2-O-ethyl-$\text{sn}$-glycero-3-phosphocholine >$\text{rac}$-1-O-octadecyl-2-O-ethylglycero-3-phosphocholine = 1-O-hexadecyl-2-O-methyl-$\text{sn}$-glycero-3-phosphocholine >$\text{rac}$-1-O-dodecyl-2-O-ethyl-glycero-3-phosphocholine. The deacetylated compound, 1-O-hexadecyl-2-lyso-$\text{sn}$-glycero-3-phosphocholine, and 1-O-hexadecyl-2,2-dimethylpropanediol-3-phosphocholine were inactive. The active analogs selectively desensitized the response to each other in the neutrophils. It is suggested that these compounds may activate cells through interaction with a stereospecific receptor.     

Effects of depurination on the fidelity of DNA synthesis.

The effect of depurination of polynucleotide templates on the fidelity of DNA synthesis in vitro has been determined. The fidelity of DNA synthesis with Escherichia coli DNA polymerase I, avian myeloblastosis virus DNA polymerase and human placenta DNA polymerase-β is decreased as a result of depurination of the poly[d(A-T)], poly[d(G-C)]and poly[d(A)]templates. The error rate with poly[d(A-T)]increased from $\text{1}\text{17,500}$ to $\text{1}\text{2100}$ using E. coli Pol I, and from $\text{1}\text{4100}$ to $\text{1}\text{1500}$ using the myeloblastosis virus DNA polymerase. Depurination of poly[d(A)]increased the error rate from $\text{1}\text{21,000}$ to $\text{1}\text{6500}$ using E. coli Pol I, and from $\text{1}\text{19,300}$ to $\text{1}\text{6100}$ using the DNA polymerase-β from human placenta. Depurination of poly[d(G-C)]resulted in an increase in the error rate with E. coli Pol I from $\text{1}\text{9200}$ to $\text{1}\text{2200}$, and with the virus DNA polymerase from $\text{1}\text{2400}$ to $\text{1}\text{1300}$. This misincorporation is shown to be directly proportional to the extent of depurination. Deletion experiments and alkaline sucrose gradient analyses suggest that the incorporation of complementary and non-complementary nucleotides is dependent on polymerization, and occurs in the same newly synthesized product. Kinetic studies and nearest-neighbor analyses indicate that the incorporation of non-complementary nucleotides occurs randomly as single-base substitutions. The nearest-neighbor studies also suggest that any of the four deoxynucleotides can be incorporated opposite apurinic sites. The number of each nucleotide incorporated relative to the number of apurinic sites was determined to be $\text{1}\text{490}$ for dGTP, $\text{1}\text{115}$ for dCTP, $\text{1}\text{2·5}$ for dATP and $\text{1}\text{1·7}$ for dTTP with both the poly[d(A-T)] and poly[d(A)] templates. The frequencies of misincorporation relative to the number of apurinic sites with the poly[d(G-C)]template were $\text{1}\text{230}$ for dATP, $\text{1}\text{120}$ for dTTP, $\text{1}\text{2·4}$ for dGTP and $\text{1}\text{1·8}$ for dCTP. Hydrolysis at the apurinic sites by alkali treatment reversed the effects of depurination on fidelity. The error rates with the depurinated templates were reduced to within 2% of those obtained prior to depurination, providing additional evidence that the misincorporation after depurination results from apurinic sites on the template. These results suggest a possible relationship between depurination of DNA and errors in DNA replication and/or repair.     

Serum glutathione S-transferases: Perinatal development,sex difference,and effect of carbon tetrachloride administration on enzyme activity in the rat

Glutathione $\text{S}$-transferase activity was determined in rat, rabbit, and guinea pig serum using styrene 7,8-oxide (SO) and benzo (a) pyrene 4,5-oxide (4,5-BPO) as substrates. Of the species tested, rat had the highest transferase activity (62.5 and 3.2 nmol/min/ml serum for SO and 4,5-BPO, respectively) and rabbit had the lowest activity. Glutathione $\text{S}$-transferase activity was 60% higher in serum from male rats than in female rats. In rats, serum enzyme specific activities (nmol/min/mg protein) were less than 1% of hepatic enzyme activities with SO, 4,5-BPO, 1,2-dichloro-4-nitrobenzene (DCNB), and 1-chloro-2,4-dinitrobenzene (DNCB). Glutathione $\text{S}$-transferase activity was also determined in rat serum during perinatal development. Serum from rats at 18 days of gestation or from 1- and 4-day-old animals had barely detectable transferase activity. Activity increased with age and reached a maximum in 140-day-old animals. The intraperitoneal administration of diethyl maleate (DEM) (0.8 ml/kg) or L-methionine-DL-sulfoximine (MS) (200 mg/kg) to male rats had no effect on serum or hepatic glutathione $\text{S}$-transferase activities 2 or 26 hr after dosing. Treatment with carbon tetrachloride (CCl₄) (1 m1/kg) caused an 11-fold increase in serum transferase activity and a 40% decrease in liver specific activities 24 hr after administration.     

Detection of sequence heterology by use of the N. Crassa nucleases

We have used the single-strand specific nucleases of $\text{Neurospora crassa}$ to detect sequence divergencies between two similar DNA molecules: restriction endonuc lease $\text{Eco}$RI produced linears from Simian Virus 40 and a variant of human origin, DAR. Enzyme treatment of the heteroduplex DNA resulted in specific cleavage into two fragments of one-third and two-thirds genome length. These two viral DNAs therefore have at least one region of heterology located about 0.35 map units from the $\text{Eco}$RI site. Due to the known specificities of the $\text{N.crassa}$ nucleases, this technique is applicable to detect mutations in RNA or DNA genomes.