A model for genetic analysis of programmed gene expression as reflected in the development of membrane antigens.

The red blood cell antigen determined in mice by the H-2^b allele was detectable by hemagglutination at birth, H-2^b-early (bE), in strain $\text{C57BL}\text{10}$, whereas that determined by H-2^a not until 3 days later, H-2^a-late (aL), in strain A. Each F₁ individual was both aL and bE. Timing genes segregated in the F₂ and BC₁ generations to give H-2^a-early (aE) and H-2^b-late (bL) in combinations with aL and bE in apparent Mendelian ratios. In some strain combinations in which H-2 alleles and strain background had been interchanged, timing type was transferred along with H-2, whereas in others it was the background which seemed to be more important for the timing type. Moreover, no recombinant types were found in the F₂ and BC₁ generations of these lines. Lines originating from the (B10 × A)F₂ generation were selected for reversed parental-strain-type phenotypes under a full-sib mating regime. True breeding aE or bL lines were not realized until the ninth to eleventh inbreeding generation. We propose a remote-cis-effect model consisting of one temporal gene tightly linked with H-2 and at least two others with special cis and recognition properties linked with each other but not with H-2.     

Synthesis of 3,4-13C2 steroids

A-ring enollactones $\text{1a}$, $\text{1b}$ or $\text{9}$ derived from 4-cholesten-3-one, testosterone benzoate or 3-oxo-4-estren-17β-yl benzoate were condensed with [1,2-¹³C₂]acetyl chloride to give intermediates $\text{2a}$, $\text{2b}$ or $\text{10}$. $\text{2a}$ and $\text{2b}$ were cyclized by acid or base to give 3,4-¹³C₂-labeled 4-cholesten-3-one and testosterone, respectively. [3,4-¹³C₂]4-Cholesten-3-one was converted via reduction of its trimethylsilyl enol ether to [3,4-¹³C₂]cholesterol. Acetyl enollactone $\text{10}$ was cyclized in acetic acid to [3,4-¹³C₂]3-oxo-4-estren-17β-yl benzoate followed by aromatization and hydrolysis to produce [3,4-¹³C₂]estradiol-17β. Alternatively, cyclization of $\text{10}$ with base afforded [3,4-¹³C₂]3-oxo-4-estren-17β-ol directly, which was then oxidized and aromatized to yield [3,4-¹³C₂]estrone. Ozonolysis of progesterone, conversion to the diketal ester $\text{16}$ and acylation followed by acid hydrolysis furnished [3,4-¹³C₂]progesterone.     

Analysis of chlorophyll fluorescence induction curves in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea as a function of magnesium concentration and NADPH-activated light-harvesting chlorophyll ab-protein phosphorylation

The effect of Mg²⁺ concentration and phosphorylation of light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ on various chlorophyll fluorescence induction parameters of isolated pea thylakoids has been studied. (1) Lowering the Mg²⁺ concentration from 3 to 0.4 mM decreases only the variable fluorescence (F_v) and the area above the induction curve while at the same time increasing the slow exponential component of the rise (β_max). (2) A further decrease in Mg²⁺ concentration from 0.4 to 0 mM decreases the initial (F₀) fluorescence level such that the ratio $\text{F}{}_{\mathrm{<mtext>v</mtext>}}\text{F}{}_{\mathrm{<mtext>m</mtext>}}$ increases slightly as does the area above the induction curve and β_max. (3) Thylakoid membranes, phosphorylated at 5 mM Mg²⁺, show an equal decrease in F_v and F₀, no change in the area above the induction curve and an increase in β_max. At 2 mM Mg²⁺, however, phosphorylation induced a more extensive quenching of F_v so that the $\text{F}{}_{\mathrm{<mtext>v</mtext>}}\text{F}{}_{\mathrm{<mtext>m</mtext>}}$ ratio was lowered and the area above the induction curve decreased while β_max increased. (4) When phosphorylated membranes were subsequently suspended in an Mg²⁺-free medium the effect on F₀ due to phosphorylation was found to be additive to that due to the absence of Mg²⁺. The effect of membrane phosphorylation on fluorescence is discussed in relation to the control of excitation energy distribution and shows that different mechanisms operate depending on the background Mg²⁺ levels. At high Mg²⁺ the phosphorylation seems to affect the absorption cross-section of Photosystem II while at lower Mg²⁺ levels there is an additional effect of increased spillover from Photosystem II to I.     

Isolation of a 110 000 molecular weight protein in the purification of the nuclear chick intestinal 1,25-dihydroxyvitamin D-3 receptor

A single protein band of molecular weight 110 000 has been obtained after sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified 1,25-dihydroxyvitamin D-3 (1,25-(OH)₂D-3) receptor from crude nuclear extracts of chick intestinal mucosa, prepared in the presence of the protease inhibitors phenylmethylsulfonyl fluoride and ?-aminocaproic acid. The nuclear extract was subjected to a six-step purification scheme, involving polymin P and ammonium sulfate fractionation, DNA-cellulose affinity chromatography, Sephacryl S-200 gel filtration, blue dextran-Sepharose and a final DNA-cellulose chromatographic step. The receptor was obtained in about 1% yield and was purified approx. 3700-fold from the nuclear extract, as assessed by specific activity. Single peaks were observed with ³H-1,25-(OH)₂D-3-labeled crude nuclear extracts on Sephacryl S-200 gel filtration ($\text{Strokes′ radius}\text{= 35.5}\text{A}\text{?}$) and sucrose density gradient centrifugation (3.5 S). Although the identity of the M_r 110 000 protein will remain inconclusive until methods for further characterization are available, it may represent evidence for a higher molecular weight form of the 1,25-(OH)₂D-3 receptor than that observed previously.     

18,19-Dihydroxydeoxycorticosterone; A novel product of cytochrome P-45011β-catalyzed reaction

After incubating 18-hydroxydeoxycorticosterone (18-OH-DOC) with cytochrome P-450_11β in the reconstituted system, the products were analyzed with HPLC. There appeared two product-peaks on the chromatogram, one of which was identified as a peak of 18-hydroxycorticosterone (18-OH-B), an expected product of the 11β-hydroxylation. Another peak did not coincide with those of any known corticoids. This unidentified product was further purified, and the purified material was analyzed by gas chromatography-mass spectrometry ($\text{GC}\text{MS}$). The mass spectrum showed that the unidentified product is one of the structural isomers of 18-OH-B. A further analysis with ¹H-NMR spectrometry indicated that a proton resonance peak of 19-CH₃ in 18-OH-DOC disappeared in the product and the methyl group of the substrate seemed to be converted to -CH₂OH. These results suggested that the unidentified product generated from 18-OH-DOC by P-450_11β-linked hydroxylase system may be 18,19-dihydroxydeoxycorticosterone (18,19,21-trihydroxypregn-4-ene-3,20-dione; 18,19-diOH-DOC), a hitherto unreported corticoid.     

In vivo biosynthesis of -linolenic acid in plants

[1-¹⁴C]acetate was readily incorporated into unsaturated fatty acids by leaf slices of spinach, barley and whole cells of $\text{Chlorella}\text{pyrenoidosa}$ and $\text{Candida}\text{bogoriensis}$. In these systems the [¹⁴C] label in newly synthesized oleate and linoleate was approximately equally distributed in the C_1–9 and the C_10–18 fragments obtained by reductive ozonolysis of these acids, whereas in a-linolenic acid over 90% of the total [¹⁴C] was localized in the C_1–9 fragment. While [1-¹⁴C]oleic acid was converted by whole cells of $\text{Chlorella}$ to [1-¹⁴C]linoleic and [1-¹⁴C]linolenic acids, [U-¹⁴C]oleic acid yielded [U-¹⁴C]linoleic acid but a-linolenic acid was labeled only in the carboxyl terminal carbon atoms. When spinach leaf slices were supplied with carboxyl labeled octanoic, decanoic, dodecanoic, tetradecanoic and octadecanoic acids, only the first three acids were converted to a-linolenic acids while the last two acids were ineffective. Thus we suggest that (a) linoleic acid is not the precursor of a-linolenic acid and (b) 12:3(3, 6, 9) is the earliest permissible trienoic acid which is then elongated to a-linolenic acid.     

Heterozygosity for the IVS-I-5(G→C) mutation with a G→A change at codon 18 (Val→Met; Hb Baden) in cis and a T→G mutation at codon 126 (Val→Gly; Hb Djonburi) in trans resulting in a thalassemia intermedia

We have analyzed the hemoglobins of a young German patient with β-thalassemia intermedia and of his immediate family and included in these studies an evaluation of possible nucleotide changes in the β-globin through sequencing of amplified DNA. One chromosome of the propositus and one of his father's carried the $\text{G}\text{T}\text{G}\text{→}\text{G}\text{G}\text{G}$ mutation at codon 126 leading to the synthesis of Hb Dhoburi or α₂β₂126(H₄)Val→Gly; this variant is slightly unstable and is associated with mild thalassemic features. His second chromosome and one of his mother's had the common IVS-I-5 (G→C) mutation that leads to a rather severe β⁺-thalassemia and the $\text{G}\text{TG}\text{→}\text{A}\text{TG}$ mutation at codon 18, resulting in the replacement of a valine residue by a methionine residue. This newly discovered β-chain variant, named Hb Baden, was present for only 2–3% in both the patient and his mother. This low amount results from a decreased splicing of RNA at the donor splice-site of the first intron that is nearly completely deactivated by the IVS-I-5 (G→C) thalassemic mutation. The chromosome with the codon 18 ($\text{G}\text{TG}\text{→}\text{A}\text{TG}$) and the IVS-I-5 (G→C) mutations has thus far been found only in this German family; analysis of 51 chromosomes from patients with the IVS-I-5 (G→C) mutation living in different countries failed to detect the codon 18 ($\text{G}\text{TG}\text{→}\text{A}\text{TG}$) change.     

Role of Mg2+ ions in the subunit structure and membrane binding properties of bacterial energy transducing ATPase.

ATPase extracted from $\text{Streptococcus faecalis}$ membranes was purified by preparative slab gel electrophoresis in the presence of Mg⁺⁺ (plus Mg²⁺ ATPase) and without Mg²⁺ (minus Mg²⁺ ATPase). The subunit composition and membrane binding capacity of both preparations was then examined. The plus Mg²⁺ ATPase had 5 types of subunits (αβγδ?) and reattached normally to depleted membranes. The minus Mg²⁺ ATPase had the αβγ and ? chains, but no δ chain, and failed to reattach to membranes. These data indicate that Mg²⁺ or a similar cationic ligand anchors the δ chain to the core enzyme complex and that the δ chain in turn is needed for membrane attachment. For the plus Mg²⁺ ATPase the data are consistent with the subunit stoichiometry and arrangement, (α₃β₃ γ ?)-Mg²⁺)_n?(δ).     

2H- and 31P-NMR studies of bilayers composed of 1-acyllysophosphatidylcholine and fatty acids

1-Palmitoyllysophosphatidylcholine has been mixed in equimolar amounts with specifically deuterated palmitic acid and the structural properties of the lipid/water phase have been studied by ²H- and ³¹P-nuclear magnetic resonance. The order profile of the free palmitic acid is very similar to that of the parent compound $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero-3-phosphocholine}$ at temperatures above the gel-to-liquid crystal phase transition. The bending of the $\text{sn-2}$ chain which is typical for diacyl lipids is not observed for the free palmitic acid. The mixture of lysolipid and palmitic acid exhibits well-defined quadrupole splittings even at temperatures below the gel-to-liquid crystal phase transition. Hence it is possible for the first time to establish an order profile in the gel-state of the lipid bilayer phase. Between carbon atoms 5 to 12 the palmitic acid chain is found to assume the extended all-trans conformation with a very small contribution from gauche defects. Towards the methyl terminal a distinct increase in the gauche probability can be noted. The motion of the phosphocholine headgroup was also studied by ²H- and ³¹P-NMR using selectively deuterated 1-palmitoyllysophosphatidylcholine. The headgroup has a considerably larger motional freedom in the mixture of lysolipid and palmitic acid than in $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero-3-phosphocholine}$. In addition, the average headgroup conformations are also different in the two systems.     

Microbial transformations of natural antitumor agents. 23. conversion of withaferin-A to 12β- and 15β-hydroxy derivatives of withaferin-A.

Microbial transformation experiments were conducted with the antitumor lactone withaferin-A. $\text{Cunninghamella elegans}$ NRRL 1393 transformed withaferin-A ($\text{1a}$) to 15β-hydroxywithaferin-A ($\text{2a}$) and 12β-hydroxywithaferin-A ($\text{3a}$). The hydroxylated metabolites were isolated by solvent extraction and were purified by column and thin-layer chromatography. Structures of the hydroxylated metabolites were determined by protonand carbon-13 NMR, IR and mass spectral analyses, and by the preparation of acylated derivatives. Compounds $\text{2a}$ and $\text{3a}$ inhibited the growth and biochemical functions of $\text{in vitro}$ grown P-388 lymphocytic leukemic cells.     

Laser-light scattering investigation of the micellar properties of gangliosides

The micellar properties of gangliosides in water solutions were investigated by quasielastic light scattering measurements. G_M1 and G_D1a gangliosides were isolated from calf brain, purified to more than 99% and dissolved in 0.025 M Tris—HCI buffer (pH 6.8) at 37°C. The average intensity of scattered light and the intensity correlation function were measured by an apparatus including a 5145 Å argon laser and a real-time digital correlator. The scattered intensity data allowed the derivation of an upper limit to the critical micelle concentration (c₀) and the evaluation of the molecular weight (M) of the micelle. The intensity correlation function gave the diffusion coefficient D, and hence the hydrodynamic radius R_H, and also contained information on the polydispersity of the sample. We find c_o < 1 × 10^?6 M for both G_M1 and G_D1a, M = 532 000 ± 50 000 and $\text{R}{}_{\mathrm{<mtext>H</mtext>}}\text{= 63.9 ± 2}\text{A}\text{?}$ for G_M1, and M = 417 000 ± 40 000 and $\text{R}{}_{\mathrm{<mtext>H</mtext>}}\text{= 59.5 ± 2}\text{A}\text{?}$ for G_D1a. The mixture 3:1 of the two gangliosides gave intermediate values for all examined parameters. The presence of cations, within the physiological concentration range. and, in particular of Ca²⁺, did not influence significantly the values of c_o and the main features of the micelle.     

Purification and nucleoside composition of a Q* - containing aspartic acid tRNA from Drosophila.

One form of aspartic acid tRNA from $\text{Drosophila}$,$\text{melanogaster}$ (tRNA_2δ^Asp) is selectively bound to columns of Con A-Sepharose. Unlike the other Q-containing tRNAs of $\text{Drosophila}$, it therefore appears that tRNA^Asp contains the more highly modified nucleoside, Q¹ (mannose form) in its anticodon. This is further supported by the chromatographic insensitivity of tRNA_2δ^Asp to NaIO₄ treatment. Utilizing Con A-Sepharose chromatography, tRNA_2δ^Asp from $\text{Drosophila}$ was purified and its nucleoside composition determined by chemical tritium labelling. In addition to the major nucleosides, this tRNA contains rT, hU, m⁵C, ψ, and Q¹, but no other modified nucleosides. Its nucleoside composition is very similar to yeast tRNA^Asp.     

Structural and functional organization of the photosystems in spinach chloroplasts. Antenna size,relative electron-transport capacity,and chlorophyll composition

The structural and functional organization of the spinach chloroplast photosystems (PS) I, II_α and II_β was investigated. Sensitive absorbance difference spectrophotometry in the ultraviolet (?A₃₂₀) and red (?A₇₀₀) regions of the spectrum provided information on the relative concentration of PS II and PS I reaction centers. The kinetic analysis of PS II and PS I photochemistry under continuous weak excitation provided information on the number (N) of chlorophyll (Chl) molecules transferring excitation energy to PS II_α, PS II_β and PS I. Spinach chloroplasts contained almost twice as many PS II reaction centers compared to PS I reaction centers. The number N_α of chlorophyll (Chl) molecules associated with PS II_α was 234, while N_β = 100 and N_{PS I} = 210. Thus, the functional photosynthetic unit size of PS II reaction centers was different from that of PS I reaction centers. The relative electron-transport capacity of PS II was significantly greater than that of PS I. Hence, under light-limiting green excitation when both Chl a and Chl b molecules are excited equally, the limiting factor in the overall electron-transfer reaction was the turnover of PS I. The Chl composition of PS I, PS II_α and PS II_β was analyzed on the basis of a core Chl a reaction center complex component and a Chl $\text{a}\text{b-}\text{LHC}$ component. There is a dissimilar Chl $\text{a}\text{b-}\text{LHC}$ composition in the three photosystems with 77% of total Chl b associated with PS II_α only. The results indicate that PS II_α, located in the membrane of the grana partition region, is poised to receive excitation from a wider spectral window than PS II_β and PS I.     

Evidence for a glucosyl-enzyme intermediate in the beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside

The reaction of almond β-glucosidase with $\text{p}$-nitrophenyl-β-$\text{D}$-glucoside has been investigated over the temperature range +25° to ?45° using 50% aqueous dimethyl sulfoxide (DMSO) as solvent. At temperatures below those at which turnover occurs a “burst” of $\text{p}$-nitrophenol proportional to the enzyme concentration is observed. Such a “burst” suggests the existence of a glucosyl-enzyme intermediate whose breakdown is rate-limiting, and provides a method for measuring the active-site normality. At pH 5.9, 25°, the presence of 50% DMSO causes an increase in K_m from 1.7×10^?3M (0%) to 1.7×10^?2M, whereas V_max is unchanged. The DMSO thus apparently acts as a competitive inhibitor with K_i = 0.7M. The Arrhenius plot for turnover is linear over the accessible temperature range with E_a = 23.0 ± 2.0 kcal/mole.     

Enzymatic formation of (20R, 22R)-20,22-dihydroxycholesterol from cholesterol and a mixture of 16O2 and 18O2: random incorporation of oxygen atoms

[4-¹⁴C]Cholesterol was incubated with an adrenocortical preparation in the presence of ¹⁶O₂ and ¹⁸O₂ devoid of significant ¹⁶O¹⁸O. Isolated (20R,22R)-20,22-dihydroxycholesterol was converted to a trimethylsilyl derivative and analyzed by gas chromatography - mass spectrometry to determine the isotope distribution of the oxygen atoms at C-20 and C-22. The ions of $\text{m}\text{e}$ 289, 291, and 293 (comprising the C₈ C-20 to C-27 side-chain and containing, respectively, ¹⁶O₂, ¹⁶O¹⁸O, and ¹⁸O₂) exhibited a binomial distribution indicating that the oxygen atoms of the vicinal glycol were drawn at random from the atomic pool of the oxygen molecules. If both side-chain hydroxyl groups had originated from the atoms of the same oxygen molecule, the ion of $\text{m}\text{e}$ 291 would have been absent.     

Trichinella spiralis: genetic basis for differential expression of phase-specific intestinal immunity in inbred mice

Responsiveness of mouse strains after phase-specific immunization with Trichinella spiralis is compared. Two strains ($\text{NFR}\text{N, NFS/N}$) showed strong overall responsiveness. The response type could be characterized in phase-specific terms as: strongly anti-adult, weakly to moderately anti-preadult, and strongly antifecundity. By comparison, congenic mice of the C₅₇B1 $\text{10}\text{Sn}$ background (B10·A, B10·D₂, B10·S, B10·Q) displayed poor total responses that could be characterized as: weakly anti-adult, very weakly anti-preadult, weakly anti-fecundity after preadult immunization, and mixed (weak and strong) after adult immunization. The $\text{C}{}_3\text{H}\text{HeJ}$ mouse appeared to be intermediate between the B10·BR and the $\text{NFR}\text{N}$ strains in overall responsiveness. Genetic determinants of anti-preadult or anti-adult responses of $\text{NFR}\text{N}$ strain mice were dominant over their B10 congenic counterparts as shown in F₁, crosses of $\text{NFR}\text{N}$ × B1O·BR mice. Since the $\text{NFR}\text{N}$ (predominantly H-2^q) and the $\text{NFS}\text{N}$ (H-2^S) are both strong responders, while the B10·Q(H-2^q) and B10·S (H-2^S) are weak, it is suggested that the major genes controlling anti-preadult and anti-adult responses are not linked to the major histocompatibility complex. However, variations in anti-adult immunity and anti-fecundity in the B10 congenic mice (B10·Q and B10·S are the strongest responders) suggest that minor genes linked to the MHC exert some control over these responses. Some evidence was obtained for gene complementation as the F₁ cross of $\text{NFR}\text{N}$ and $\text{NFS}\text{N}$ mice responded more vigorously than the parental lines. We conclude that multiple genes determine anti-T. spiralis intestinal responses in mice. The major genes are unlinked to the major histocompatibility complex whereas several minor genes are linked.     

Interactions and space requirement of the phosphate head group of membrane lipids: The single crystal structures of a triclinic and a monoclinic form of hexadecyl-2-deoxyglycerophosphoric acid monohydrate

The crystal structures of a triclinic form (HPA1) and a monoclinic form (HPA2) of hexadecyl-2-deoxyglycerophosphoric acid monohydrate were determined by single crystal analysis. The unit cell dimensions for HPA1 are $\text{a = 4.75, b = 5.72, c = 44.36}\text{A}\text{?}$ and α = 91.0, β = 101.5, γ = 100.5° (P$\text{1}$) and for HPA2, $\text{a = 4.75, b = 5.72, c = 88.72}\text{A}\text{?}$ and γ = 100.8° (P2₁). In both structures the molecules are fully extended and pack tail-to-tail in bilayers with tilting (47°) hydrocarbon chains. In HPA2, however, the chain tilt alternatingly changes direction in adjacent bilayers, giving rise to a doubled unit cell which spans two bilayers. The dihydrogen phosphate groups interact by hydrogen bonds and are arranged in rows. Laterally between these phosphate rows the water molecules are accommodated producing a compact two-dimensional network of hydrogen bonds. The packing cross-section in the layer plane of the dihydrogen phosphate monohydrate group is 26.7 Ų in both structures. The hydrocarbon chains pack according to the triclinic (T|) chain packing mode. In HPA2, however, the chain packing is somewhat less compact with accounts for a 2% increase in the molecular volume. In both structures the ether oxygen is accommodated into the hydrocarbon matrix without distortion of the chain packing.     

Kinetic parameters for the binding of p-nitrophenyl alpha-d-mannopyranoside to concanavalin A.

Binding of the chromogenic ligand p-nitrophenyl α-d-mannopyranoside to concanavalin A was studied in a stopped-flow spectrometer. Formation of the protein-ligand complex could be represented as a simple one-step process. No kinetic evidence could be obtained for a ligand-induced change in the conformation of concanavalin A, although the existence of such a conformational change was not excluded. The entire change in absorbance produced on ligand binding occurred in the monophasic process monitored in the stopped-flow spectrometer. The value of the apparent second-order rate constant (k_a) for complex formation (k_a = 54,000 s^?1m^? at 25 °C, pH 5.0, Γ/2 0.5) was independent of the protein concentration when the protein was in the range of 233–831 μm in combining sites and in excess of the ligand. The apparent first-order rate constant (k_?a) for dissociation of the complex was obtained from the rate constant for the decomposition of the complex upon the addition of excess methyl α-d-mannopyranoside (k_?a = 6.2 s^?1 at 25 °C, pH 5.0, Γ/2 0.5). The ratio $\text{k}{}_{\mathrm{<mtext>a</mtext>}}{}_{\mathrm{<mtext>?</mtext><mtext>a</mtext>}}$ (0.9 × 10₄m^?1) was in reasonable agreement with value of 1.1 ± 0.1 × 10⁴m^?1 determined for the equilibrium constant for complex formation by ultraviolet difference spectrometry. Plots of ln($\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{T}$) and ln($\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{T}$) vs $\text{1}\text{T}$ were linear (T is temperature) and were used to evaluate activation parameters. The enthalpies of activation for formation and dissociation of the complex are 9.5 ± 0.3 and 16.8 ± 0.2 kcal/mol, respectively. The unitary entropies of activation for formation and dissociation of the complex are 2.8 ± 1.1 and 1.3 ± 0.7 entropy units, respectively. These entropy changes are much less than those usually associated with substantial changes in the conformation of proteins.