Structural studies on the freezing-point-depressing protein of the winter flounder Pseudopleuronectes americanus

The freezing-point-depressing protein from the winter flounder, $\text{Pseudopleuronectes americanus}$ has been shown from circular dichroism measurements to possess a large proportion (～85%) of the α-helical conformation in aqueous solution (pH 8.0) at ?1°C. The helical content decreases as the temperature is raised. Viscosity data at ?1°C indicate an asymmetric shape for the protein molecule compatible with its high helical content. Thus, the secondary and tertiary structure of this freezing-point-depressing protein as well as its primary structure (reported elsewhere), are found to be different from its counterpart glycoproteins isolated from the Antarctic fish.     

RNA synthesis in Escherichia coli during K + -depletion

During K⁺ depletion of a mutant of $\text{Escherichia}$$\text{coli}$ which cannot concentrate this cation, protein synthesis is inhibited but RNA formation continues. The RNA produced during K⁺ depletion was analyzed by gel electrophoresis. It was found that 4S, 5S and 23S RNA were synthesized by K⁺-depleted cells whether uninfected or infected with phage T4. In addition, an RNA species moving close to 16S (presumably 17S) and material of about 6–10S were made during K⁺ depletion. These species of RNA were not evident in growing cells. Methylation of RNA is severely inhibited during K⁺ depletion.     

A manganese-stimulated endonuclease from Bacillus subtilis

An endonuclease activity has been identified in extracts of $\text{Bacillus subtilis}$. This activity is stimulated by Mn⁺⁺ or Ca⁺⁺ ions but not by Mg⁺⁺ ions. The enzyme catalyzes the breakdown of native DNA of high molecular weight to fragments of molecular weights ranging from 3 × 10⁶ to 20 × 10⁶. A variety of DNA's from sources such as $\text{B. subtilis, Salmonella}$ and T7 phage are attacked. About 61% of the activity of the cells is released into the medium during protoplast formation under conditions where 98% of the glucose 6-P dehydrogenase activity is retained by the cells.     

The early synthesis and possible function of a 0.5 X 10(6) Mr RNA after transfer of dark-grown Spirodela plants to light.

A 0.5 × 10⁶$\text{M}$_r RNA found in plastids of the aquatic angiosperm $\text{Spirodela}$, is synthesized at a much higher rate than any other rapidly labeling RNA species about $\text{3–3}\text{1}\text{2}$ h after dark-grown plants are transferred to light. The pulse labeling kinetics of the 0.5 × 10⁶$\text{M}$_r RNA after transfer to light, argue against its involvement in the biogenesis of plant rRNAs. Although poly(A) RNA is found in $\text{Spirodela}$, poly(A) sequences are not detected in the 0.5 × 10⁶$\text{M}$_r RNA; yet a sucrose gradient fraction which includes RNA of this $\text{M}$_r stimulates amino acid incorporation by an $\text{E. coli}$ cell free extract more than other RNA fractions. The possible involvement of the 0.5 × 10⁶$\text{M}$_r RNA as a chloroplast messenger is discussed.     

An Escherichia coli K12 mutant carrying altered ribosomal protein (S10).

An $\text{Escherichia coli}$ mutant (JE14373) carrying decreased stability of stable RNA species was found to have altered electrophoretic mobility of a 30S ribosomal protein (S10). Recombinants covering $\text{str}$ gene (76 min on $\text{E. coli}$ linkage map by Bachmann, Low and Taylor, 1976 (ref. 1)) obtained from a cross of CSH64 × JE14373, restored normal S10 protein. The size analysis of RNAs labeled for 15 min with [³H]uridine showed 50 to 60 % decrease of 16S RNA in this mutant strain, but almost no decrease of 23S RNA at 10 or 40 min after addition of rifampicin. On the other hand, no change was observed in the stability of both rRNA pieces in its parental PA3092 strain even at 40 min after addition of rifampicin.     

The synthesis of proteolipid protein by isolated rat liver mitochondria

About 15% of the total (³H)leucine incorporated into protein by isolated rat liver mitochondria $\text{in}\text{vitro}$ could be extracted by chloroform:methanol. This incorporation was inhibited by chloramphenicol and carbomycin, both specific inhibitors of mitochondrial protein synthesis. SDS-gel electrophoresis of the mitochondrial membrane revealed 6–7 labeled bands. Label in the proteolipid fraction was present mainly in a band of 40,000 molecular weight. Several labeled bands observed in gels of the mitochondrial membrane were not removed or changed by extraction with chloroform:methanol suggesting that some, but not all, of the proteins synthesized by rat liver mitochondria are proteolipids.     

Loss of hepatoma ribosomal RNA during warfarin therapy

40–50% decreases in cytoplasmic ribosomal RNA were observed in mouse hepatoma implants, but not livers, after 4–5 daily warfarin injections. Similar treatment greatly depressed rates of $\text{in}\text{vivo}$¹⁴C-orotate incorporation into hepatoma ribosomal RNA in the cytoplasm. Labelling of mature 18S and 28S RNA in the nucleus appeared to be unaffected. Possible mechanisms for this warfarin effect are briefly discussed.     

Carcinogen-protein complexes in mammary gland after administration of 3-methylcholanthrene

Twenty-four hrs after i.p. administration of ³H-3-methylcholanthrene (³H-MCA) to 8–10 weeks old virgin or 14 days pregnant $\text{BALB}\text{c}$ mice, the macromolecules and ³H-hydrocarbon-protein complexes in their mammary cytosols were extensively resolved according to molecular size. The profiles of the macromolecules at both stages of mammary development were generally similar, and contained at least five families of molecular size of modes: > 300,000, 181,000, 88,000, 44,000 and 27,000 daltons. At least three species of ³H-hydrocarbon-protein complex were present. The principal complex had a modal molecular weight of 83,000, and the two minor species > 300,000 and 47,000. Only a small amount of complex similar to the principal species was produced $\text{in vitro}$ by incubation of ³H-MCA with cytosol at 1–4°, indicating that the carcinogen is probably metabolized prior to interaction $\text{in vivo}$ with its principal target protein.     

Molecular characterization of a stable Flac plasmid

F$\text{lac}$S is a thermostable extrachromosomal element isolated in $\text{Salmonella typhimurium}$ which is altered in its replication as compared to its precursor F_ts114$\text{lac}$. Sedimentation of both these plasmids in alkaline sucrose gradients has indicated a difference in their sizes. Contour length measurements of open circular plasmid DNA molecules photographed in the electron microscope have revealed the estimated molecular weight of F_ts114$\text{lac}$ to be 81 × 10⁶ daltons while that of F$\text{lac}$S is 109 × 10⁶ daltons. F$\text{lac}$S may carry a segment of $\text{S. typhimurium}$ chromosomal or cryptic plasmid DNA.     

Regulation of protein synthesis in rabbit reticulocyte lysates: Separation of heme regulated protein kinase into a high and a low molecular weight species

Protein synthesis in rabbit reticulocyte lysates is regulated by heme. In heme deficiency, a heme regulated protein kinase (HRI) is activated that phosphorylates initiation factor eIF-2. Consequently, eIF-2 is inactivated. Results described in this report show that HRI exists in crude and highly purified preparations in two forms; a high molecular weight component which sediments at a sedimentation co-efficient of 14–15S and a previously described 5.8S component (Ranu, R. S. and London, I. M. (1976) $\text{Proc}\text{.}\text{Natl}\text{.}\text{Acad}\text{.}\text{Sci}\text{.}\text{USA}$ 73, 4349–4353). The 14–15S HRI selfphosphorylates poorly and undergoes dissociation into the 5.8S component via an intermediate of 8.5–9S. The 5.8S HRI, on weight basis, is about 5–10 times more active than the 14–15S HRI. In addition, a phosphoprotein phosphatase has been detected in lysates that dephosphorylates selfphosphorylated HRI. This observation suggests that phosphate on HRI turns over. These findings may be relevant ot the mechanism of activation and inactivation of HRI in the absence and presence of heme $\text{in}\text{situ}$.     

Monotertiarybutylhydroquinone effects on Tetrahymena pyriformis.

The molecular toxicity of monotertiarybutylhydroquionone (TBHQ) was studied using $\text{Tetrahymena}$$\text{pyriformis}$ as a model cell system. TBHQ at 26 ppm in the media inhibited cell growth by 50%. TBHQ inhibited the oxidation of ¹⁴C-acetate to ¹⁴CO₂. In addition, increasing concentrations of TBHQ decreased the incorporation of ¹⁴C-acetate into lipids and protein, ¹⁴C-amino acids into protein, ³H-uridine into RNA and ³H-thymidine into DNA. The incorporation of ¹⁴C-acetate into glycogen increased with concentrations up to 20 ppm TBHQ in the media while glycogen synthesis decreased with 40 ppm TBHQ.     

N-6-methyl-adenosine in adenovirus type 2 nuclear RNA is conserved in the formation of messenger RNA

In the biogenesis of adenovirus type 2 messenger RNAs, methylation occurs at the 5′ end (cap) and to internal adenosine residues to yield N⁶-methyl-adenosine (m⁶A) (Sommer et al., 1976; Moss &; Koczot, 1976; Wold et al., 1976). The kinetics of accumulation of ³H from methyl-labeled methionine and ¹⁴C from uridine into Ad-2²-specific RNA was measured late in Ad-2 infection. As reported previously (Nevins &; Darnell, 1978a), the rate of accumulation of [¹⁴C]uridine label in nuclear RNA was approximately four- to fivefold faster than in the cytoplasmic RNA, indicating a conservation of about 20% for the total RNA. The initial rates of [³H]methyl label in m⁶A in nuclear RNA and in the cytoplasmic RNA were approximately equal, suggesting a complete (or nearly complete) conservation of m⁶A.In accord with the accumulation kinetics, the ratio of ³H to ¹⁴C was higher in cytoplasmic RNA than in nuclear RNA that hybridized to equivalent regions of the Ad-2 DNA.A mathematical model was designed to evaluate the accumulation of methyl label in m⁶A, taking into consideration the three major parameters that affect the accumulation curves: equilibration of the S-adenosyl-methionine pool, the nuclear dwell time of sequences destined to be mRNA, and the cytoplasmic stability of mRNA. The half-time ($\text{t}\text{1}\text{2}$) for pool equilibration was determined experimentally to be 22 minutes and the nuclear dwell time and the mean life-time of cytoplasmic mRNA were estimated from ¹⁴C label to be about 30 and 70 minutes, respectively.The model gave an excellent fit to the data when the $\text{t}\text{1}\text{2}$ for pool equilibration time of 24 ± 2 minutes, a nuclear dwell time of 25 ± 10 minutes, and a mean cytoplasmic mRNA life-time of 75 ± 30 minutes were used to evaluate accumulation curves. Even when data from a restricted region of the genome, $\text{40.5–52.6}$, which encodes the main portion of at least five 3′ co-terminal mRNAs whose spliced junction with the tripartite leader sequence varies from $\text{38, 40, 43, 45}$, and $\text{48}$ was analyzed, it appeared that m⁶A was conserved.Finally, m⁶A was found to be added in a brief label (3.5 min) mainly to nuclear molecules that were longer than any cytoplasmic RNA. The conservation of m⁶A and its addition prior to splicing raise the possibility that internal methylations are involved, in the formation of mRNA.     

Isolation and partial characterization of a mucin-type glycoprotein from plasma membranes of human melanoma cells

Plasma membranes were isolated from HM7 melanoma cells grown in the presence of [³H]glucosamine and Na₂³⁵SO₄ or [³H]mannose and [¹⁴C]glucosamine. The labelled glucoconjugates were solubilized with 0.6 M lithium diiodosalicylate/0.5% Triton X-100. Fractionation of glycoconjugates by repeated chromatography on columns of Sepharose CL-6B and DEAE-Sepharose and by affinity chromatography on WGA-Sepharose yielded three radiochemically homogenous glycoproteins. One of these having an apparent molecular weight of 100 000 was found to contain clusters of (AcNeu)_{1 or in2} å [Gal å GalNAc] linked $\text{O-}\text{glycosidically}$ to the protein. One other glycoprotein contained both $\text{O-}\text{glycosidically}$ and $\text{N-}\text{glycosidically-linked}$ oligosaccharides, and the third contained only $\text{N-}\text{glycosidically-linked}$ carbohydrates. Preliminary results indicate that the 100 000 molecular weight mucin-type glycoprotein is present in significantly reduced quantities in cultured human fetal uveal melanocytes. Further, the bulk of the glycoproteins from the melanocytes were of lower molecular size compared to those from the melanoma cells.     

Characterization of an alpha-glucan from chick embryo sternum whose synthesis is stimulated by L-3,5,3'-triiodothyronine and human serum.

Incorporation of [³H]glucose into macromolecular components of 12-day chick embryo sternum incubated in vitro was stimulated by both human serum and l-3,5,3′-triiodothyronine. Under all conditions, 65–70% of the radioactivity was incorporated into glycosaminoglycans. About 10% of the radioactivity was incorporated into a fraction separable by ion-exchange chromatography which was stimulated two- to sixfold by addition of 2–10 nm triiodothyronine and 5–20% ($\text{v}\text{v}$) human serum. Further characterization of this fraction by paper electrophoresis at pH 3.5 showed the presence of two components, one apparently anionic and one neutral. All of the increase in incorporation of [³H]glucose was into the former species. Acid hydrolysis of this material showed that it contained only glucose. Treatment with α-amylase released 78% of the label as maltotriose and maltose; digestion with crystalline β-amylase released 75% as maltose; and treatment with glucoamylase and α-amylase released 93% as glucose. There was no incorporation of any amino acid into this fraction, nor could any incorporation of [³²P]phosphate, [³⁵S]sulfate, [³H]uridine, or [³H]acetate be demonstrated. Mild acid hydrolysis (0.1 N HC1, 100 °C, 10–20 min) converted the material to a neutral species with a much lower molecular weight. The results indicate that chick embryo sternum contains a species of glycogen whose synthesis is stimulated by thyroid hormones and other serum factors.     

Translation of AKR-murine leukemia viral RNA in an E. coli cell-free system

High molecular weight RNA isolated from the oncogenic type C murine leukemia virus, AKR-MuLV, stimulates the incorporation of radioactive amino acids into protein in an $\text{E. coli}$ cell-free system. Analysis of the translational products by SDS polyacrylamide gel electrophoresis demonstrated the synthesis of at least three proteins corresponding in molecular weight to several authentic viral proteins. Positive immunoprecipitation tests also confirm the translational product as AKR-MuLV related. Although at least 18 proteins were found on analysis of disrupted murine leukemia virions, only three were synthesized $\text{in vitro}$ in response to AKR-MuLV RNA in the $\text{E. coli}$ cell-free system.     

Synthesis and biological activities of pseudopeptide analogues of LH-RH: agonists and antagonists

Using solid phase methods, seven agonist and antagonist analogues of LH-RH have been prepared containing enzyme-resistant CH₂S linkages as selected amide bond replacements. Agonists modified at the 5–6, 6–7 and 9–10 position had 2, < 0.1, and 10% of the $\text{in}\text{vitro}$ activity of LH-RH, respectively. Among potential antagonists, 6–7 position analogues showed only minimal inhibitory activity but N- and C-terminal modified analogues retained substantial LH-RH-LH and FSH inhibitory activity. In addition, a 1–2 position methylene thioether analogue of the parent [Ac-Pro¹, D-Phe², D-Trp^3,6]LH-RH antagonist was completely inhibitory at 30 ng $\text{in}\text{vitro}$ and represents the first such structure-modification that may be at least as active as its corresponding amide linked congener. However, neither 1–2 nor 9–10 methylene thioether position antagonists showed $\text{in}\text{vivo}$ antiovulatory activity at the 250 μg level.     

Dimers of escherichia coli F' factors

Covalently closed circular DNA dimers of several $\text{E.}$$\text{coli}$ sex factors have been isolated. One of these, F′451, a dimer of F′450, has a molecular weight of $\text{ca.}$ 230 × 10⁶ daltons. F′451 (λ) containing a λ prophage has a molecular weight of 260 × 10⁶ and is probably the largest covalent closed circle of DNA yet reported. These dimers arise spontaneously and are of unknown origin and significance.     

The mechanism of action of initiation factor 3 in protein synthesis. II. Association of the 30S ribosomal protein S12 with IF-3

Dimethylsuberimidate was used to crosslink ¹⁴C-labeled chain initiation factor 3 to $\text{E. coli}$ 30S particles. The crosslinked ribosomal proteins were analyzed by dodecyl sulfate polyacrylamide gel electrophoresis, and one major radioactive aggregate was found corresponding to a molecular weight of 41,000. Ribosomal protein S12 was identified to be crosslinked to IF-3 by immunological cross-reactivity.