Accumulation of immunoglobulin messenger ribonucleic acid in immunized mouse spleen.

We have measured the concentration of mRNAs coding for immunoglobulins, k and lambda type light chains and gamma 1 type heavy chain, in mouse spleen cells activated by bacterial lipopolysaccharide or sheep red blood cells. These mRNAs were quantitated by hybridization to radioactive DNA complementary to highly purified immunoglobulin mRNAs from mouse myelomas. In the lipopolysaccharide-stimulated spleen cells, only light chain mRNA accumulated, whereas gamma 1 type heavy chain mRNA remained unvaried. The light chain mRNA concentration also increased in purified bone-marrow-derived lymphocytes. The lipopolysaccharide-induced light chain mRNA was similar to light chain mRNAs purified from myelomas. The accumulation and disappearance of light chain mRNA in bone-marrow-derived lymphocytes coincide with the kinetics of synthesis of immunoglobulin M which is the major species induced by lipopolysaccharide. In sheep red blood cell stimulated spleen, the specific accumulation of k type light chain and gamma 1 type heavy chain mRNAs parallels immunoglobulin G synthesis. These results seem to indicate that the increment of immunoglobulin mRNA concentration in bone-marrow-derived lymphocytes is important for induction of immunoglobulin synthesis.     

Regulated transport of messenger ribonucleic acid from isolated liver nuclei by nucleic acid binding proteins

Rat liver nucleocytosolic messenger ribonucleic acid (mRNA) transport is shown to be regulated by proteins with a high affinity for nucleic acids. In the cell-free system described, the energy-dependent transport of all RNA classes [transfer RNA (tRNA), mRNA, and ribosomal RNA (rRNA)] exhibited a dependence upon the availability of discrete minor sets of cytosol proteins. In addition to having a different level of saturation, only the mRNA "transport protein" activities are increased by adenosine cyclic 3',5'-phosphate (cAMP), an effect most likely mediated by a cAMP-dependent protein kinase. The mRNA transport proteins were isolated from cytosol by precipitation with streptomycin sulfate followed by deoxyribonucleic acid (DNA)-cellulose affinity chromatography, or from oligo-(thymidylate)-cellulose bound cytoplasmic messenger ribonucleoprotein (mRNP) particles by high-salt extraction. Either method yielded a protein fraction which exhibited a 1000-fold increase in mRNA transport activity as compared to cytosol. Over one-half of the mRNA transport activity is associated with the mRNP of the cell. A partial homology between the cytosol and mRNP-derived proteins was demonstrated by polyacrylamide gel electrophoresis. One major (20 000 daltons) and several minor proteins (23 000, 52 000, 54 000, and 72 000 daltons) were in common. Nuclear 4-5S exited from in vitro incubated nuclei in three phases, according to their differential in vivo rates of labeling and intranuclear pool sizes. The amount of nuclear RNA transported in vitro as mRNA (about 1.0%) agrees wtih the in vivo estimates. Additional evidence for in vivo equivalence was provided by the physicochemical characterization and bioassay of the RNA. The transported mRNA sedimented in urea-sucrose gradients as an 8-18S heterodisperse product. This RNA initiated cell-free translation with the synthesis of precursor peptides as diverse in size as those for albumin and alpha 2U-globulin. The relative abundancies of various transported mRNAs were different than the corresponding abundancies of liver cytoplasmic mRNAs.     

Structure of the constant and 3' untranslated regions of the murine Balb/c gamma 2a heavy chain messenger RNA.

The complete sequence for the constant and 3' untranslated regions of a mouse gamma 2a immunoglobulin heavy chain mRNA is reported. The sequence is 1093 nucleotides long coding for the CH1 (amino-acids 118-214), the Hinge (215-230), the CH2 (231-340) and the CH3 (341-447). The 3' untranslated region is 103 nucleotides long preceding the poly(A). The nucleotide sequence predicts as in the case for gamma 1 and gamma 2b heavy chains an additional lysine residue before the termination codon. This sequence has been compared to the corresponding sequences of gamma 1 and gamma 2b heavy chain mRNAs. These sequences are respectively 75% and 84% homologous. The CH2 domains of gamma 2a and gamma 2b are 95% homologous at the nucleotide level. The cross-over point of a gamma 2a - gamma 2b heavy chain variant is located in a segment of 73 perfectly matching nucleotides. The 3' non coding regions of gamma 2a and gamma 2b are 89% homologous.     

Exploring sequence/folding space: folding studies on multiple hydrophobic core mutants of ubiquitin

The stability, dynamic, and structural properties of ubiquitin and two multiple hydrophobic core mutants were studied. One of the mutants (U4) has seven substitutions in the hydrophobic core (M1L, I3L, V5I, I13F, L15V, V17M, and V26L). On average, its side chains are larger than the wild-type, and it can thus be thought of as having an overpacked core. The other mutant (U7) has two substitutions (I3V and I13V). On average, it has smaller side chains than the wild-type, and it can therefore be considered to be underpacked. The three proteins are well-folded and show similar backbone dynamics (T(1), T(2), and HNOE values), indicating that the regular secondary structure extends over the same residue ranges. The crystallographic structure of U4 was determined. The final R(factor) and R(free) are 0.198 and 0.248, respectively, at 2.18 A resolution. The structure of U4 is very similar to wild-type ubiquitin. Remarkably, there are almost no changes in the positions of the C(alpha) atoms along the entire backbone, and the hydrogen-bonding network is maintained. The mutations of the hydrophobic core are accommodated by small movements of side chains in the core of mutated and nonmutated residues. Unfolding and refolding kinetic studies revealed that U4 unfolds with the highest rates; however, its refolding rate constants are very similar to those of the wild-type protein. Conversely, U7 seems to be the most destabilized protein; its refolding rate constant is smaller than the other two proteins. This was confirmed by stopped-flow techniques and by H/D exchange methodologies. This work illustrates the possibility of repacking the hydrophobic core of small proteins and has important implications in the de novo design of stable proteins.     

Gene conversion in human immunoglobulin gamma locus shown by unusual location of IgG allotypes

The constant region of the gamma 1, gamma 2 and gamma 3 heavy chains of the human IgG1, IgG2 and IgG3 immunoglobulins carries antigenic determinants or G1m, G2m and G3m allotypes, which are genetic markers of these subclasses. The exceptional presence on gamma 1 and gamma 2 chains of Gm allotypes usually located on the CH3 domain of gamma 3 shows an unexpected clustering of base changes and subsequent identity of short DNA sequences in the CH3 exon of the non-allelic gamma 1, gamma 2 and gamma 3 genes. Such clusters of substitutions are not easily explained on the classical basis of point mutations. A gene conversion, which substituted a segment of the gamma 1 or gamma 2 gene with the homologous region of the non-allelic gamma 3 gene, is more likely. Other examples of possible conversion involving the gamma genes are described. The conservation or the restoration of short sequences produced by the conversion events might be related to the biological properties of the constant region of the heavy chains.     

A matched set of rat/mouse chimeric antibodies. Identification and biological properties of rat H chain constant regions mu, gamma 1, gamma 2a, gamma 2b, gamma 2c, epsilon, and alpha

Rat C regions mu, gamma 1, gamma 2a, gamma 2b, gamma 2c, epsilon, and alpha have been characterized by means of chimeric antibody technology. A set of rat/mouse Ag-specific (anti-4-hydroxy-3-nitrophenacetyl) antibodies was constructed that differ only in the H chain constant region but carry identical V region and L chain, both of which are of mouse origin. All rat constant regions could be expressed and m.w. were as expected from the protein sequence. A slight variation in mobility within the IgG subclasses allowed us to establish a hierarchy for the sizes of the four gamma H chains; gamma 2b greater than gamma 1 greater than gamma 2c greater than gamma 2a. Rat IgG2c and IgG2b could be purified on both protein A and protein G while rat IgG2a could only be purified on protein G. Rat IgM and IgG2b were the most potent in C-mediated hemolysis. This was not simply a consequence of the amount of C1q bound because IgG2c bound C1q efficiently but was relatively poor in cell lysis. In ADCC using human effector and target cells, IgG2b and IgG1 were the most effective.     

Immunoglobulin mu- and gamma-ribonucleic acid sequences in thymocytes and splenocytes from normal and hyperimmune mice

The immunoglobulin heavy-chain ribonucleic acid (RNA) repertoire of mouse thymocytes was examined. Previously, this laboratory reported immunoglobulin alpha-chain RNA sequences in mouse thymocytes [Near, R. I., & Storb, U. (1979 Biochemistry, 18, 964]. We have extended these studies to encompass mu, gamma 2b, and gamma 1 heavy-chain RNA sequences, mu-, gamma 2b-, and gamma 1-messenger RNAs (mRNAs) were purified from myelomas to 45, 22, and 54% purity, respectively. Each of these mRNAs faithfully translated into the appropriate immunoprecipitable protein in a reticulocyte lysate translation system. The gamma 1-mRNA translated into two major immunoprecipitable products of about 52 500 and 51 000 daltons while mu- and gamma 2b-mRNAs yielded only a single major protein. Complementary deoxyribonucleic acids (cDNAs) prepared from the mRNAs were used as hybridization probes and revealed the presence of about 70 mu-RNA sequences per average thymocyte as determined by hybridization kinetics, while gamma 1 and gamma 2b sequences were at the limits of detection. The mu-RNA sequences are present in the cytoplasm and are greater than 50% polyadenylated. Upon hyperimmunization of mice with sheep red blood cells, gamma 1-RNA in splenocytes increased by about 100-fold while only slightly increasing in thymocytes. mu and gamma 2b increased 2-3-fold in splenocytes and only slightly in thymocytes. The results argue against RNA sequences appearing in thymocytes due to contamination with peripheral confirmed with cloned cDNA probes. Thymocyte RNA analyzed by Northern blots displayed bands of the same size as those in splenocyte RNA or in purified mRNA when hybridized to mu, gamma 2b and alpha cloned probes. Also, K light-chain RNAs of the same size were found in spleen and thymus by using a cloned K-DNA probe. The results are consistent with the thymus containing mu-, alpha-, and K- and small amounts of gamma 1- or gamma 2b-RNAs coding for heavy- and light-chain-like proteins which may play a role in T-cell function.     

Thermodynamics of ligand binding and denaturation for His64 mutants of tissue plasminogen activator kringle-2 domain

The contribution of His64 to the function and stability of tissue plasminogen activator (t-PA) kringle-2 domain (His244 in t-PA numbering) has been studied by using microcalorimetric methods to compare the ligand binding and thermal denaturation behavior of wild-type kringle-2 and mutants having His64 replaced with Tyr or Phe. This site was examined because modeling studies suggested that the His64 side chain could play an important role in ligand binding by forming an ion-pair with the carboxylate of the ligand, L-lysine. Kringle-2 domains were expressed by secretion of the 174-263 portion of t-PA in E. coli and purified as previously described for the wild-type domain. Both mutant proteins retain affinity for L-lysine, although reduced three- to four-fold relative to wild-type, demonstrating that His64 does not interact with the ligand carboxylate through an ion-pair interaction or by hydrogen bonding. The H64Y substitution does result in an altered specificity of the lysine binding site with the mutant domain having greatest affinity for a ligand of 6.8 A chain length, whereas the wild-type domain prefers an 8.8 A long ligand. For both wild-type and mutant, the binding of the optimal chain length ligand is dominated by enthalpic effects (delta H = -6,000 to -7,000 cal/mol) and T delta S accounts for less than 15% of delta G. In addition, the H64Y mutant differs from wild-type in the effect of ligand alpha-amino group modification on binding affinity. Based on examination of the x-ray structure recently determined for wild-type kringle-2, the specificity changes accompanying the H64Y substitution probably result from changes in side chain interactions in the lysine binding site. Thermal denaturation experiments show that the H64Y mutant is also more stable than the wild-type protein with the difference in stabilization free energy (delta delta G) equal to 2.7 kcal/mol at 25 degrees C and pH 3. The increased stability of the mutant appears to be related to the difference in hydrophobicity between His and Tyr.     

Structural relationships between human erythrocyte sialoglycoproteins beta and gamma and abnormal sialoglycoproteins found in certain rare human erythrocyte variants lacking the Gerbich blood-group antigen(s).

The human erythrocyte membrane sialoglycoproteins beta and gamma are important for the maintenance of the discoid shape of the normal erythrocyte. In this paper we show that the human erythrocyte sialoglycoproteins beta and gamma (hereafter called beta and gamma) are structurally related. Rabbit antisera produced against purified beta and beta 1 and rendered specific to the cytoplasmic portion of these proteins also react with the cytoplasmic portion of gamma. Some human anti-Gerbich (Ge) sera react with the extracellular portion of both beta and gamma. This reactivity is shown to be directed towards a common epitope on beta and gamma. However, most anti-Ge sera do not react with beta, but react with an extracellular epitope only present on gamma. All individuals who lack the Ge antigens lack beta and gamma. In some cases abnormal sialoglycoproteins are present in the erythrocytes, and these are shown to be structurally related to beta and gamma. Rabbit antisera raised against the purified abnormal sialoglycoprotein from a Ge-negative erythrocyte type reacted with the cytoplasmic portion of both beta and gamma. Unlike normal beta and gamma, the abnormal sialoglycoproteins found in Ge-negative erythrocytes migrate as a diffuse band on SDS/polyacrylamide-gel electrophoresis. Studies using endoglycosidases suggest that the diffuse nature of these bands results from carbohydrate heterogeneity and that the abnormal sialoglycoproteins contain N-glycosidically linked oligosaccharides with repeating lactosamine units. Such polylactosamine chains are not present on normal beta or gamma.     

The chemical heterogeneity of human hemoglobin F. Direct evidence for the existence of three types of gamma chain, the G gamma I, A gamma I, and A gamma T chains.

Direct evidence is presented for the existence of three types of gamma chain of human hemoglobin F. A modification of a CM-cellulose chromatographic method has allowed the incomplete separation of these gamma chains while high pressure liquid chromatography and fingerprint analyses of tryptic peptides of zones of the isolated gamma chains, and amino acid analyses of isolated peptides were used to identify the chains. These studies have shown that the presence of a glycyl residue in position 136 (G gamma chain) is directly related to that of an isoleucyl residue in position 75 (I gamma chain), thus indicating the existence of an G gamma I chain, and that the presence of an alanyl residue in position 136 (A gamma chain) can be related to that of an isoleucyl residue in position 75, thus suggesting the existence of an A gamma I chain. When the isoleucyl residue at positive 75 is replaced by a threonyl residue, invariably it is related to the alanyl substitution at position 136 (A gamma T chain). These data support indirect evidence from case analyses and family studies which were published before, and indicate that the T gamma chain is an allele of the A gamma which should be renamed the A gamma T chain.     

Pre-early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs.

The studies described here demonstrate that the expression of many early adenovirus mRNAs is dependent upon the activity of a pre-early viral product. This viral gene product is defective in adenovirus 5 host range (Ad hr) group I mutants. Adenovirus 5 host range mutants were previously isolated by their ability to replicate in the adenovirus 5-transformed human embryonic cell line 293 and by their inability to replicate efficiently in HeLa cells (Harrison, Graham and Williams, 1977). The group I complementation class of host range mutants has been mapped by marker rescue between 0 and 4.4 units (Frost and Williams, 1978). We have used the S1 nuclease gel technique to examine the expression of early mRNA after infection of HeLa cells with Ad5 hr group I and II mutants. The Ad5 hr group II mutants stimulate the synthesis of a wild-type pattern of early mRNAs. In contrast, infection of HeLa cells with Ad5 hr group I mutants gives rise to only two early mRNAs. These mRNAs map from 1.5–4.4 units, or in the same region as the Ad5 hr group I mutations. Since infection of HeLa cells with Ad5 hr group I mutants was defective for synthesis of cytoplasmic mRNAs complementary to three early regions in the right half of the genome and to the early region 4.5–11.0 units, we also analyzed nuclear RNA from these cells by the S1 nuclease gel technique for the presence of precursor RNA chains. Nuclear precursors were not detected in Ad5 hr group I-infected HeLa cells, suggesting that the gene product defective in these mutants is required for synthesis of stable nuclear RNA from the three early regions in the right half of the genome and from the early region 4.5–11.0 units.     

Escherichia coli mutants defective in the gamma subunit of proton-translocating ATPase: intracistronic mapping of the defective site and the biochemical properties of the mutants

Various hybrid plasmids carrying a portion of the gene for the gamma subunit of the H+-ATPase of Escherichia coli complemented five mutants defective in the enzyme in a genetic test, indicating that the mutants are defective in the gamma subunit. Since the nucleotide sequence of genomic DNA carried on the plasmids is known, the defective site(s) of the mutants could be located within the gene for the gamma subunit as follows: KF10 and NR70, KF1, and KF12 and KF13 have a mutation causing a defect(s) in amino acid residues 1 to 82, 83 to 167, and 168 to 287, respectively, of the gamma subunit. The biochemical properties of all these mutants except NR70 were analyzed in terms of proton permeability of the membranes and assembly of F1. Results suggested that KF1 and KF10 have defective F1 without at least the alpha and beta subunits on their membranes, whereas KF12 and KF13 have F1's of rather similar structure to that of the wild type. Attempts were made to purify F1 oF KF12 as a single complex. Although the F1 complex dissociated during purification, active alpha and beta subunits of KF12 were partially purified. On the basis of these biochemical and genetic results, it is suggested that structural alterations in the primary sequence of the gamma subunit corresponding to residues 1 to 167 cause more extensive defects in the assembly of F1 than alteration in the sequence of residues 168 to 287.     

Differential gene expression in wild-type and X-ray-sensitive mutants of Chinese hamster ovary cell lines.

Complementary DNA cloning, differential screening and Northern hybridization techniques were used to study differential gene expression in the wild-type Chinese hamster ovary (CHO) K1 cell line and its two X-ray sensitive mutants, xrs-5 and xrs-6. 11 species of mRNAs were found underexpressed in the two independently isolated mutants. The steady-state levels of those mRNAs are 3-26-fold less in the two mutants, depending on the particular species. 6 of the underexpressed mRNAs have been identified by comparing the sequences of the cloned cDNAs to the known sequences in GenBank. 4 of them code for the structural proteins of ferritin heavy chain, nonmuscle myosin light chain 3nm, ribosomal protein S17 and L7, respectively. The other two have strong homology with mouse B2 or retroviral sequences. The remaining 5 mRNAs did not show significant homology with any of the known sequences and apparently represent newly isolated species. The effect of 137Cs gamma-rays on the expression of the 11 mRNAs has been studied. Radiation inhibited the expression of the B2-like gene in the mutants but not in the wild-type CHO cells. The levels of the other 10 mRNAs were not affected by radiation. The underexpression of this group of genes in both xrs-5 and xrs-6 mutants seems to be related to their radiation-sensitive phenotype, although the specific gene responsible has not been identified. Two models are proposed to explain the mechanism of underexpression. It is suggested that a cellular factor or/and chromosome structural changes are involved.     

Use of two-dimensional protein-pattern analysis for the characterization of Arabidopsis thaliana mutants

Total proteins extracted from wild-type plants of Arabidopsis thaliana Heyhn, an etiolated mutant, a de-etiolated mutant and a mutant affected in cotyledon morphology, were analyzed by two-dimensional gel electrophoresis. Computer analysis of two-dimensional gels allowed the characterization of the mutants by a set of proteins showing a differential expression when compared with the wild-type plant grown under the same conditions. The overlap between comparisons of the diferent mutants with the wild-type allowed the identification of groups of polypeptides which, since their expression is altered in several mutants, might be involved in certain physiological functions. For example, this approach showed a possible involvement of actin in the elongation process. The simultaneous analysis of the two-dimensional protein patterns of different mutants seems, therefore, to be a promising approach to characterize proteins involved in various physiological functions.Abbreviations 2-D two-dimensional - IEF isoelectrofocusing - M_r relative molecular mass - E17.1 E17 mutant grown in light - E22.1 E22 mutant grown in light - E44.d E44 mutant grown in darkness - WT.d wild-type plants grown in darkness - WT.1 Wild-type plants grown in light We would like to thank our laboratory colleagues J.A. Traas, H. Höfte and D. Bouchez (all from INRA) for useful discussions throughout this work. Also grateful thanks to Mr Zivy (Lab. Génétique des Sytèmes Végétaux. INRA. La Ferme du Moulon, Gif-sur-Yvette, France) for helpful discussions concerning 2-D gel analysis and to I. Small (Lab. Biologie Cellulaire. INRA, Versailles, France) for English text corrections.     

Histidyl-transfer ribonucleic acid synthetase mutants requiring a high internal pool of histidine for growth

Mutants that require histidine due to an altered structural gene for the histidyl-transfer ribonucleic acid synthetase (hisS) have been isolated by a general selection for histidine-requiring strains in which the mutation producing histidine auxotrophy is unlinked to the histidine operon. One of the mutants has been shown to require an abnormally high internal histidine pool for growth owing to an altered synthetase that is unstable at low histidine concentrations. It is difficult to determine accurately the K(m) for histidine of the synthetase enzyme from the mutant because of the instability of the enzyme at limiting histidine concentrations; however, a histidine K(m) value has been estimated that is approximately 100 times higher than the histidine K(m) of the wild-type enzyme. For the mutant strains to achieve the high internal pool of histidine required for growth, all the systems that transport histidine from the growth medium must be functioning to capacity. Amino acids that interfere with histidine transport strongly inhibit the growth of the mutants. The mutants have been useful in providing a selective genetic marker for transductional mapping in the hisS region. The mutants are discussed as representative of a general class of curable mutants that have an altered enzyme with poor affinity for a substrate or coenzyme.     

Translation of embryonic-chick tendon procollagen messenger ribonucleic acid in two cell-free protein-synthesizing systems

Embryonic-chick tendon poly(A)-containing RNA was translated in the wheat-germ and mRNA-dependent rabbit reticulocyte-lysate systems. The ability of each system to synthesize polypeptides similar to pro-alpha chains of collagen was tested on the bases of electrophoretic mobility and susceptibility to highly purified bacterial collagenase. Very small amounts of polypeptides in the size range of pro-alpha chains were synthesized in the wheat-germ system, whereas efficient synthesis of two polypeptides similar to pro-alpha1 and pro-alpha2 chains was achieved in the reticulocyte lysate. The collagenous nature of the major high-molecular-weight products synthesized was demonstrated by their susceptibility to collagenase and ability to act as a substrate for purified collagen proline hydroxylase. Determinations of the relative amounts of these translation products suggest that the 2:1 ratio of pro-alpha1 and pro-alpha2 chains found in type I procollagen is reflected in proportional amounts of translatable mRNA for pro-alpha1 and pro-alpha2 chains. Comparisons of the electrophoretic mobilities of hydroxylated and unhydroxylated reticulocyte-lysate translation products were made with appropriate standards of hydroxylated and unhydroxylated procollagen polypeptides. The results suggest that, in common with a number of secreted proteins, procollagen is synthesized as pre-pro molecules consistent with the ;Signal Hypothesis'.