Experimental studies on a mutant gene (e) preventing the differentiation of eye and normal hypothalamus primordia in the axolotl

The phenotype of axolotls (Ambystoma mexicanum) homozygous for the mutant gene e (“eyeless”) is different from normal in that (1) no optic vesicles develop in $\text{e}\text{e}$ embryos, (2) $\text{e}\text{e}$ larvae from posthatching onward are darker than normal white larvae, and (3) fully grown $\text{e}\text{e}$ animals are sterile.Experiments reported here show that eyelessness in $\text{e}\text{e}$ embryos results from a direct effect of the gene on presumptive forebrain ectoderm; not on the mesoderm that induces the ectoderm to form eyes. Homotopic grafts of normal presumptive ectoderm on $\text{e}\text{e}$ blastula hosts differentiated complete eyes, but reciprocally grafted embryos were always eyeless. Similarly, grafts of either $\text{e}\text{e}$ or normal presumptive prechordal mesoderm into normal hosts gave normal eyes, but in the mutant hosts no eyes developed. Thus the e gene affects only the ectodermal component of the inductive system for eye formation.Genetically eyeless (pigmented) cells, when interspersed prior to gastrulation among genetically eyed (albino) cells in the eye preprimordium, are induced to form clones of pigmented retinal epithelium in the albino host eye.The sterility of $\text{e}\text{e}$ larvae appears also to be due to a direct effect of the e gene on the ectodermal (neural plate) primordium of the hypothalamus. Grafts of normal cells which included the hypothalamic, but not the optic or anterior pituitary primordia, always restored fertility to $\text{e}\text{e}$ recipients.The mutant pigmentation phenotype was demonstrated to be a consequence of eyelessness and, therefore, an indirect effect of the gene. The pigment pattern of normal embryos from which both optic vesicles were removed resembles that of the mutants. In addition, implantation of a single full-sized, functional eye was able to restore the normal pigmentation, but not fertility, to $\text{e}\text{e}$ recipients.     

Complementation analysis of replication and maintenance functions of broad host range plasmids RK2 and RP1

It has previously been concluded that regions tentatively designated trfA and trfB, located at 16–18.7 and 54–56 kb, respectively, on the genome of broad host range plasmid RK2 provide trans-acting functions involved in plasmid replication and maintenance in Escherichia coli (Thomas et al., 1980). A third region, the replication origin, ori_RK2, located at 12 kb on the genome, is also required. A segment of DNA containing ori_RK2 can be linked to a nonreplicating selective marker and can replicate as an autonomous plasmid so long as DNA of RK2 carrying the gene for one or more trans-acting replication functions is present in the same cell on an independent plasmid or integrated into the chromosome. It is demonstrated here that the trfA region alone can provide the trans-acting functions necessary for replication from ori_RK2. Deletion of the trfB region in trans to an ori_RK2 plasmid does not correlate with alteration in copy number or stability of the ori_RK2 plasmid. Temperature-sensitive mutants defective in plasmid maintenance can apparently arise from mutations in both the trfA and trfB regions as indicated by complementation analysis of three different mutants. The trfA and trfB regions from two mutant plasmids have been cloned and used to allow a physically separate but functionally dependent ori_RK2 plasmid to replicate at 30 °C. When the source of trfA and trfB is a trfB mutant the ori_RK2 plasmid is temperature stable but is temperature sensitive when the source is a trfA mutant. This confirms that only trfA is essential for initiation at and elongation from ori_RK2 which is probably the primary event in RK2 replication and suggests that the trfB region plays some other role in plasmid maintenance in plasmids carrying all three regions, ori_RK2, trfA, and trfB.     

A modified amino acid analyzer with greater versatility

An older amino acid analyzer was easily modified with commercially available components to update it by providing more convenient sample application, more accurate timing, greater sensitivity, and automatic regeneration. New features were introduced to permit the optional use of different buffer systems and methodologies as well as the convenient analysis of unusual basic amino acids.     

Isolation and characterization of the hydrophobic COOH-terminal domains of the Sindbis virion glycoproteins

Digestion of intact Sindbis virions with α-chymotrypsin produced a single membrane-associated peptide derived from each of the two virion glycoproteins (referred to as RE1 and RE2, or roots derived from E1 and E2, respectively). Amino acid composition data and NH₂-terminal sequence analysis established their location at the extreme COOH-terminal end of each glycoprotein. RE1 and RE2 are rich in hydrophobia amino acids and insoluble in aqueous solutions in the absence of detergents, and show differential solubility in organic solvent systems designed for the extraction of lipids. Essentially all of the covalently attached palmitic acid associated with E1 and E2 was found to be clustered in their hydrophobic, membrane-associated roots. Beginning six to seven residues from their NH₂ termini, RE1 and RE2 contain uninterrupted sequences of hydrophobic amino acids similar in terms of amino acid composition and length to the transmembrane anchors found in other bitopic integral membrane proteins. By comparing the sequence and composition data obtained here with the sequences of E1 and E2 deduced from complementary DNA sequence analysis (Rice & Strauss, 1981) we can make several observations. First, following their uncharged, putative intramembrane segments (33 and 26 amino acids, respectively), E1 and E2 contain clusters of predominantly basic amino acids. By structural analogy to known transmembrane proteins, E1 probably spans the bilayer but contains only a few residues exposed on the inner face of the virion envelope. In contrast, E2 and PE2 (the precursor to E2), which have been shown to span the bilayer completely, contain an additional 33 COOH-terminal residues, which could be either exposed on the cytoplasmic face of the lipid bilayer or which could loop back into the membrane. This region at the extreme COOH-terminal end of E2, which is protected by the virion envelope from digestion by a-chymotrypsin, contains a second uncharged domain (23 amino acids in length) whose orientation is unknown, but which may be involved in the highly specific interaction of the transmembrane glycoproteins in the plasma membrane with the cytoplasmic nucleocapsid during budding.     

Polarity of microtubules of the mitotic spindle

Microtubules are polar structures that grow preferentially at one end. Measurement of their rate of directional growth can be used as a polarity indicator to determine their orientation with respect to a nucleation site. The results are interpreted to signify that the microtubules originating from the centrosomes and chromosomes of the mitotic spindle are antiparallel to each other.     

A modified method for amino acid analysis with ninhydrin of Coomassie-stained proteins from polyacrylamide gels

Triton X-100 (from three different suppliers) and Brij 35, substituted ethers of polyoxyethylene alcohols, were found to contain variable amounts of powerful oxidizing impurities representing a range of 0.04-0.22% H₂O₂ equivalents. These detergents contain also a considerable quantity of carbonyl compounds (0.5-2%) originating from carboxylic acids and ketones or aldehydes. Tween 20, also a polyoxyethylene detergent, and sodium dodecyl sulfate were free from oxidizing contamination. Aqueous solutions of Triton X-100 and Brij 35 (1–4%) reacted readily with SH groups of protein and nonprotein molecules as well as with Fe²⁺ ion. Both detergents were purified from the oxidizing impurities by treating aqueous solution of detergent with either NaHSO₃ or SnCl₂ followed by an extraction procedure. The present findings may clarify as well as complicate the interpretation of previous studies where these detergents were used for biological purposes, especially in enzyme and protein purifications, or when present in assay procedures that are based on the formation or consumption of reducing reagents.     

Interaction of Mg+2 with beef heart mitochondrial ATPase (F1).

The soluble mitochondrial ATPase, F₁, can be slowly inactivated by incubation with Mg⁺² in a manner consistent with the observations of Moyle and Mitchell $\text{(}\text{FEBS}\text{Lett}\text{.}\text{56}$, 55 (1975)). This inhibition results in a low initial rate of ATP hydrolysis upon addition to an ATPase assay medium of F₁ which has been incubated with Mg⁺². This inhibition, however, is completely reversible by Mg·ATP in a time dependent process and results in the rate of ATP hydrolysis increasing during the ATPase assay to reach control levels after 30 sec. The length of the lag is independent of the F₁ concentration in the ATPase assay and the lag is also completely reversed by subsequent incubation with excess EDTA before assay.F₁ is unstable if incubated with EDTA in the absence of free nucleotides or Mg⁺². The rate of inactivation increases with decreasing protein concentration until a limiting rate is reached at high dilution. Mg⁺² in excess of the EDTA or 50 μM ADP stabilize the F₁ against the inactivation but cannot reverse prior denaturation.     

Fractionation and molecular-weight determination of large polypeptides by gel filtration in guanidiniun chloride

The interaction of several N-acetyl-d-glucosamine analogs and of sialyl lactose with the lectin wheat germ agglutinin was studied by nuclear magnetic resonance. N-²H₃-acetyl-d-gluocosamine was synthesized and found to displace the N-acetyl methyl signal toward its free chemical shift in N-acetylglucosamine and N-acetylneuraminic acid demonstrating common binding sites for the latter two compounds. The N-acetyl methyl signal of the α-methylglucoside of N-acetylglucosamine could be titrated but a 3-deoxy analog could not, the latter exhibiting very weak binding and demonstrating the importance of the 3-OH group in the binding process. Sialyl lactose (an N-acetylneuraminic acid analog) was rather tightly bound to the lectin. N-F₃-acetyl-d-glucosamine was synthesized and its binding to the lectin was studied at pH 4, 4.5, 5.1 by ¹⁹F NMR. The two anomers were found to bind with nearly equal K_d′s but exhibited a pH and anomer dependent Δ (total bound chemical shift). The -CF₃ analog was found to bind considerably stronger to the lectin than the -CH₃ compound. The clear resolution of the α and β anomers of this molecule make it a very useful probe of the lectin binding site.     

Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA

The vaccinia virus genome is a single, linear, duplex DNA molecule whose complementary strands are naturally cross-linked. The molecular weight has been determined by contour length measurements from electron micrographs to be 122 ± 2.2 × 10⁶. Denaturation mapping techniques indicate that the nucleotide sequence arrangement of the DNA is unique. Two forms of cross-linked vaccinia DNA were observed in alkaline sucrose gradients. The relative S-values of the two cross-linked species were appropriate for a single-stranded circle and a linear single strand, each with a molecular weight twice that expected for an intact, linear, complementary strand of vaccinia DNA. The fraction of sheared vaccinia DNA able to “snap back” after denaturation suggested a minimum of two crosslinks per molecule. Full-length single-stranded circles were observed in the electron microscope after denaturation of vaccinia DNA. Partial denaturation produced single-stranded loops at the ends of all full-length molecules. Exposure of native vaccinia DNA to a single strand-specific endonuclease isolated from vaccinia virions caused disruption of the cross-links, as assayed by alkaline sedimentation, and produced free single-strand ends when partially denatured DNA was observed in the electron microscope. We conclude that vaccinia DNA contains two cross-links, one at or near (within 50 nucleotides) each end in a region of single-stranded DNA. Two models for the cross-links are presented.     

A method using 3-O-methyl-D-glucose and phloretin for the determination of intracellular water space of cells in monolayer culture.

A method is presented for determining the extent of methylation of tRNAs synthesized in mammalian and bacterial cell systems and is based upon determining the distribution of radioactivity associated with the guanine constituents of total cellular tRNA preparations previously labeled with [2-¹⁴C]guanosine and with [methyl]-³H or -¹⁴C]methionine. Whereas labeling with guanosine provides a means of assessing the extent of methylation of the [2-¹⁴C]guanine residues incorporated into tRNA, methionine labeling provides a measure of the percentage of [methyl-³H or -¹⁴C]methylated constituents that are methylated guanines. Analyses such as the above reveal that the tRNA of KB cells acquires approximately three times as many methyl groups as that of E. coli B tRNA. Coupled with the knowledge that both mammalian and bacterial tRNA preparations contain an average of 24 guanine residues per molecule, the above analyses further reveal that 7.2 and 2.4 methyl groups are incorporated into each tRNA molecule synthesized in exponentially growing KB- and E. coli B-cells, respectively. Additional information regarding the extent of formation of individual methylated constituents per tRNA molecule synthesized is presented.     

Quantification of l-glutamine using Escherichia coli glutamate synthase: A sensitive fluorometric assay

A simple, sensitive fluorometric assay for l-glutamine is described. l-Glutamine is quantified by measuring the amount of NADPH oxidized in the presence of a glutamine-containing sample (2–20 nmol), 5 mm α-ketoglutarate, 1 mm EDTA, 63 μM NADPH, and purified Escherichia coli glutamate synthase (EC 2.6.1.53). High concentrations of l-glutamate and ammonia do not interfere. The method has been applied to quantification of l-glutamine in serum.     

The combined determination of indolyl-3-acetic and abscissic acid in plant materials

A method for isoelectric focusing of antibodies in agarose gels with ampholytes synthesized in the laboratory from pentaethylenehexamine is presented. The ampholytes are easy to prepare, give results comparable to those with commercial ampholytes, and are much less expensive. Substituting agrarose bonded to plastic film for the polyacrylamide gels on glass plates commonly used offers many advantages and enhances the usefulness of isoelectric focusing as a tool for studying antibody molecules.     

Pairing of inosine with adenosine in codon position two in the translation of polyinosinic acid

Poly (I) codes for valine even more than for glycine. Since the ratio is constant over a wide range of Mg²⁺ concentrations the ambiguity appears to be inherent in the translation of I-I-I, rather than induced by the relatively high Mg²⁺ concentration (or the streptomycin) required with this messenger. These findings indicate that I in codon position 2 can pair readily not only with C (complementing glycine codon GG_⁵) but also with A (complementing valine codon GU_).I in anticodon position 3 is also known to pair with A (as well as with U and C). Crick's model for this pair would require a large increase in interstrand distance, rather than simply “wobble”. This stretch would be avoided in an alternative model, suggested by Sakore &; Sobell (1969), in which A would rotate from the usual anti to the syn conformation. Since a large stretch would seem difficult to accommodate in the less flexible middle codon position the present findings suggest that the rotation model for I·A pairing merits further exploration.     

Quantitative determination of amino acid racemization in heat-alkali-treated melanotropins: Implications for peptide hormone structure-function studies

The treatment of frog skins (in vitro) and frogs (in vivo) with melanotropins that have been heated briefly in aqueous alkali resulted in prolonged skin darkening. It has been postulated that this increase in melanotropic activity is related to the partial racemization of amino acid residues of the melanotropins. Quantitative determination of the extent of racemization of eight amino acids (Val, Pro, Met, Phe, Glu, Asp, Nle, Ser) present in α-melanotropin (α-MSH), [4-norleucine]-α-MSH, β_porcine-melanotropin (β_p-MSH), and [7-norleucine]-β_p-MSH after brief heat-alkali treatment, was accomplished using a high-resolution gas chromatographic technique. Phenylalanine-7 in α-MSH and [4-norleucine]-α-MSH and phenylalanine-10 in β_p-MSH and [7-norleucine]-β_p-MSH were found to be partially racemized to a greater extent than expected. Other amino acid residues were also racemized to unexpected degrees. The subsequent synthesis of an α-MSH analog containing d-phenylalanine-7, [4-norleucine, 7-d-phenylalanine]-α-MSH, resulted in a highly potent melanotropin with ultralong biological activity, as determined by frog skin bioassay, stimulation of mouse melanoma cell tyrosinase activity, and activation of mouse melanoma adenylate cyclase.     

Characterization of glycosaminoglycans from normal and fluoride treated rabbit iliac crest

Glycosaminoglycans from rabbit iliac crest was isolated and characterised. Glycosaminoglycans isolated from iliac crest reveals the presence of chondroitin sulphate A, chondroitin sulphate C and hyaluronic acid. However, glycosaminoglycans isolated from fluoride treated rabbit iliac crest shows the presence of dermatan sulphate (or chondroitin sulphate B) in addition to the above mentioned components. Significance of the appearance of dermatan sulphate in response to fluoride treatment is discussed.     

Multiple acid phosphatases in avian pectoral muscle--the postmicrosomal supernatant acid phosphatase is elevated in avian dystrophic muscle

There are at least three forms of acid phosphatase in avian pectoralis muscle differing in molecular weight, subcellular location, and response to various substrates and inhibitors. These enzymes are separated by differential sedimentation into postmicrosomal supernatant, lysosomal, and microsomal activities with apparent molecular weights in Triton X-100 of 68,000, 198,000, and 365,000, respectively. All of the enzymes show acid pH optima (pH approximately 5), but the postmicrosomal supernatant form is distinctly different from the other two forms in its resistance to most common phosphatase inhibitors and in its reduced activity against several organic phosphates. Quantitation of these three forms of acid phosphatase in normal and dystrophic avian pectoralis muscle shows that the postmicrosomal supernatant form is significantly elevated in dystrophic muscle; at 33 days ex ovo, 84% of the increased acid phosphatase activity in dystrophic muscle can be attributed to the postmicrosomal supernatant form. The microsomal form is only slightly elevated; the level of the lysosomal form is not altered.     

Failure of preparative flat bed electrofocusing to resolve rat liver chromosomal proteins

Rat liver nonhistone chromosomal (NHC) proteins were preparatively fractionated by isoelectrofocusing in 40 × 15 × 0.5-cm layer of Sephadex G75-8 m urea gel. The fractions were collected and analyzed by reclectrofocusing and by SDS-polyacrylamide gel electrophoresis. It is shown that most of the rat liver NHC proteins do not focus as single bands but rather within a wide pH interval. The conclusion is drawn that a protein mixture as complex as the total chromatin protein can not be completely resolved by isoelectrofocusing because the protein molecules form equilibrium complexes either with the ampholines or among themselves.     

Histamine H2-receptors in the gastric mucosa: role in acid secretion.

Currently, two major hypotheses dominate thinking about the role of histamine in the regulation of gastric acid secretion. Code has proposed that histamine is the final common mediator of secretagogue action on the parietal cell while Konturek and Grossman have suggested a multi-receptor control of the secretory process. Experimental results derived from the use of recently synthesized histamine H₂-receptor antagonists have been used by both groups to support their hypotheses. Paradoxically, these hypotheses depend on the presumed specificity of the H₂-antagonists in blocking histamine mediated acid secretion while the apparent lack of such secretagogue specificity of the H₂-antagonists is an important basis for the development of the hypotheses. Our review will analyze the experimental evidence which implicates the histamine H₂-receptor in the control of hydrogen ion secretion as well as evidence for and against receptor specificity in the gastric mucosa of histamine H₂-receptor antagonists.