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... One method of experimentally testing these theories is to determine the number of polynucleotide sequences in a genome that are complementary to immunoglobulin mRNA. Workers in several laboratories (4,5,10,13,20,26,27,30,31) have reported interesting experiments regarding the reiteration frequency of immunoglobulin genes. They have normalized the experimental data of DNA-RNA hy- ...
... When the annealing time is t1,..., ti,..., tn, let us assume the proportion of the mRNA remaining in the single-stranded form is xh..., xi,..., xn. From formula , if we let  and the parameter w is known, we can calculate the truth residuals defined as follows,  However, these truth residuals cannot be calculated because w is unknown. However, if we assume a reasonable starting value "w°" instead of the parameter w by some graphical or theoretical methods, we can obtain the computation residuals as follows:  The problem is to get a further improved value for the parameter w using the data (xi, ti), w° and Zi. ...
... H is defined as follows :  We do this by differentiating H with respect to Ow and setting the derivative equal to zero.  From equation , we obtain  Since ...
The polyribosomes synthesizing γ-globulin have been isolated by the achievement of specific precipitation using bentonite-treated anti-IgG antibody. The RNA extracted from the immunochemically precipitated polysomes was tested for its ability to direct the synthesis of proteins in a cell-free system. The specific γ-globulin-synthesizing activity (cpm of γ-globulin synthesized/μg RNA) of this RNA was 10-fold greater than that from total polysomes. γ-globulin mRNA (messenger RNA) isolated by immunoprecipitation was more than 89% pure with respect to contamination by other species of mRNA. The products synthesized by the cell-free system were also analyzed by sodium dodecyl sulphate(SDS)-polyacrylamide gel electrophoresis.
This RNA has been hybridized with mouse myeloma DNA. The estimation of immunoglobulin gene reiteration was carried out using hybridization kinetics with consideration given to the DNA/RNA ratio since the estimation from the “half Cot value” is not accurate. The results suggest that in the mouse there are about 20 copies per subgroup of genes coding for the variable region of the H and L chains.
... Several attempts have been made to characterizr immunoglobulin genes by means of hybridization kinetics at DNA excess (103)(104)(105)(106). This type of measurement is of exceptional interest in the case of immunogiobulin genes since the strict germ line theory of antibody diversity predicts that the constant region of immunoglobulin chains should be coded for by repeated DNA sequences, whereas the variable region should be coded for by less highly repeated sequences. ...
... Also, estimates of the degree of repetition of these sequences range from 40 copies per genome (103) to 5,000 copies per genome (105). Workers in three laboratories concluded that the results are consistent with the germ line theory of antibody diversity (103,105,106), but in one laboratory that the results are consistent with the somatic theory (104). Unfortunately, there are uncertainties about the purity of the immunogtobulin mRNA preparations used by the various investigators and also about the extent to which mRNAs other than immunoglobulin mRNAs are transcribed from repeated sequences. ...
The past several years have seen a virtual revolution in the study of eukaryotic mRNA. Among the notable recent achievements are the positive identification of mRNA precursors in HnRNA, the enumeration of the DNA sequences from which mRNA is transcribed, and the finding that mRNA in cultured cells is much more stable than was previously believed. One of most far-reaching discoveries has been the finding that mRNA in eukaryotes contains poly A. This discovery, aside from providing a powerful tool for mRNA isolation, has generated a large body of research into the properties and metabolism of poly A itself. In addition, the finding of a poly A-associated protein has given a renewed stimulus to the study of proteins associated with mRNA. This review is devoted to a discussion of these and related achievements, and some of their implications
... The probe used contains, therefore, the entire V and C sequences, as well as the untranslated regions (9,10). So far, there have been two limitations of such studies: (i) the lack of control of the fate of the [125I]mRNA probe during the course of hybridization and the possibility that the observed extensive RNA breakdown could be nonrandom; and (ii) the finding of two distinct transitions in the hybridization profile, corresponding to components with different gene reiteration frequencies (11)(12)(13)(14)(15)(16)(17). In this paper, an extension of preliminary reports (18), we present data obtained under hybridization conditions allowing the entire sequence of the L-chain mRNA probe, including, therefore, the V region, to remain available for hybridization throughout the reaction. ...
... A first transition in the hybridization kinetic curve (Fig. 3), analogous to that observed with 14S mRNA, has been reported for various preparations of L-chain mRNA (11)(12)(13)(14)(15), as well as for fractions containing Ig heavy chain mRNA (16,17). With a single exception (16), this rapidly hybridizing RNA corresponds to DNA sequences reiterated 200 to 300 times. ...
RNA fractions rich in immunoglobulin light (L)-chain mRNA were isolated from mouse myeloma MOPC 41 by procedures previously described, and chemically labeled with 125I. These RNA fractions were hybridized with MOPC 41 DNA under conditions of DNA excess. Hybridization conditions were chosen under which the entire sequence of the L-chain mRNA probe, thus including the variable region, remains available for hybridization throughout the reaction. The hybridization (C0t) curve showed double transition kinetics, with one component corresponding to about 250 gene copies and the other to about two to four copies. In contrast, when MOPC 41 L-chain mRNA was further purified as a single band by gel elecptrophoresis in 99% formamide, the hybridization curve showed only a single transition, corresponding to about two to four genes, with the disappearance of the "reiterated" component. That component resulted therefore from contaminating RNA species. The data indicate that no reiteration can be detected by RNase or by hydroxylapatite for the genes corresponding to the entire sequence of MOPC 41 L-chain mRNA, including the untranslated segments, within the limits of detectability of short reiterated segments. It thus appears that there is only one or very few genes corresponding to the 41 L-chain variable region "subgroup" in MOPC 41 DNA. The possibility that the variable genes of plasmocytes might result frm a combination of several nonreiterated germline genes is discussed.
When a foreign substance is introduced into the body, the organism responds by synthesizing relatively large amounts of immunoglobulins which will react specifically with the immunogen. Originally, this was thought to involve a relatively straightforward series of events in which antigens were processed by phagocytic cells, plasma cell precursors representing selected clones were activated by specific antigens, and then cell proliferation and differentiation led to the production of specific antibodies. However, within this model it was not clear how recognition of self, tolerance, and the availability of precursor cells ready to make every possible antibody was achieved. Although these and other difficult questions have still not been resolved, it has become clear that between the initial introduction of antigen and the activation of the plasma cell to produce antibodies, the body carries out a highly complex series of events involving a multiplicity of signals mediated through soluble factors and the direct and indirect interactions of multiple cell types. Subpopulations of thymus (T) derived and bone marrow (or in the bird, bursa of Fabricius) (B) derived lymphocytes interact with macrophages and with each other to produce negative (suppressive) or positive (collaborative) responses. Genes are activated. Some cells are stimulated to replicate and differentiate whereas others are prevented from responding. All this requires what Jerne (1974) has called a “network of information” and is so complex that the details are only just beginning to be discussed.
Hybridization kinetic analyses with synthetic DNA indicate that there are only two to three copies of the kappa constant region gene per haploid genome. This result lends weight to the argument that the immunoglobulin light chain is encoded by more than one continuous gene sequence.
The in vitro iodination of nucleic acids is employed in a wide diversity of applications, such as molecular hybridization experiments at both the molecular and cytological levels, nucleotide sequencing analysis by fingerprinting methods, and nucleic acid secondary structure analysis by iodination of non-base-paired cytosine residues. Under optimal conditions, the primary features of the nucleic acid iodination technique include (1) permitting the incorporation of relatively large amounts of a covalently bound, highly radioactive isotope into the molecular structure of nucleic acids and (2) avoiding extensive degradation or other deleterious alteration of the nucleic acid as a consequence of the labeling procedure. This chapter describes the role of 125I in molecular hybridization experiments. 125I is a highly advantageous isotope for use in molecular hybridization experiments. The principal advantage of 125I is its intrinsically high specific radioactivity because of its relatively short 60-day half-life. Because of this fact, it is possible to prepare labeled nucleic acids of high specific radioactivity by the introduction of limited quantities of 125I atoms. The incorporation and retention of iodine atoms in stable linkage with nucleic acids is strongly affected by (1) the pH of the reaction, (2) the ionic strength, (3) the nucleic acid concentration, (4) the time and temperature of the reaction, (5) the ratio of cytosine to iodide, (6) the concentration of iodide, and (7) the reaction termination step.
We have synthesized and characterized cDNA complementary to purified mRNA derived from the lambda chain producing myeloma tumor, RPC-20. This cDNA is of sufficinet length to encode the constant region and a major portion of the variable region sequence of the lambda gene. In addition, the expected range of cross-hybridization of this lambda probe has been shown to extend to several different members of the closely related lambda subgroup, as well as to a member of the lambda subgroup represented by MOPC-315. Since there are a minimum of seven known members of the common lambda subgroup in addition to MOPC-315, these sequences, in accordance with the germ line hypothesis, must be represented by a minimum of eight variable region genes. Using the RPC-20 cDNA probe and hybridization kinetic analysis, this sequence was found to be represented as approximately two copies per haploid genome in DNA derived from a variety of k-and lambda-producing tumors and normal tissue. Inasmuch as the cross-hybridization range of the probe has been assessed and a minimum size of the lambda subgroup determined, this observation tends to rule out separate germ line genes corresponding to each individual lambda light chain variant. Certain reservations about these conclusions are discussed.
32P-labeled light chain messenger RNA was prepared from mouse MOPC 21 myeloma cells. The messenger RNA was hybridized to purified repetitive nuclear DNA and both the hybridized (repetitive 32P-RNA) and nonhybridized (nonrepetitive 32P-RNA) fractions were isolated. Only the nonhybridized RNA gave a T1 ribonuclease fingerprint showing oligonucleotides derived from the variable and constant regions of the light chain messenger RNA. In addition, this fingerprint showed oligonucleotides derived from the untranslated regions of the light chain messenger RNA. The nonrepetitive 32P-RNA was shown to rehybridize only with the unique fraction of total nuclear DNA. The rapidly hybridizing part of the unfractionated 32P-RNA preparation, therefore, is not a component of the light chain messenger RNA itself. Complementary DNA was prepared with reverse transcriptase using unlabeled light chain messenger RNA as template, and the transcripts were fractionated into various size classes. Complementary DNA molecules greater than 900 bases in length hybridized with both the initial messenger RNA and with the nonrepetitive 32P-RNA but failed to hybridize with excess purified repetitive 32P-RNA. The rapidly hybridizing component of the messenger RNA fraction, therefore, does not appear to be transcribed by reverse transcriptase. It is concluded that, under the experimental conditions used, the light chain messenger RNA hybridizes exclusively with unique DNA.
A procedure is described for the isolation of highly purified heavy-chain immunoglobulin mRNAs from a variety of mouse plasmacytomas (IgA, IgG, and IgM producers). The use of fresh tissue and the rapid isolation and direct extraction of membrane-bound polyribosomes were found to be essential in obtaining large quantities of undegraded heavy-chain mRNAs. The individual mRNAs were purified by two cycles of oligo(dT)-cellulose chromatography, sodium dodecyl sulfate-sucrose gradient centrifugation, and electrophoresis on 98% formamide containing polyacrylamide gels. When added to a cell-free protein-synthesizing system from wheat germ, the MPC-11 γ2b and H2020 α heavy-chain mRNAs efficiently directed the synthesis of a predominant product of 55,000 molecular weight, while the synthesis of a 70,000 dalton protein in addition to other lower molecular weight polypeptides were observed with MOPC 3741 μ mRNA. All of these proteins were immunoprecipitable with class-specific heavy-chain antisera, and in the case of the γ2b in vitro products good correspondence in a comparative trypsin-chymotrypsin fingerprint with in vivo labeled γ2b heavy chain was observed. The γ2b and α heavy-chain mRNAs possessed a chain length of ~1800 nucleotides and the μ mRNA a size of ~2150 nucleotides when examined under stringent denaturation conditions. The purities of the α, γ2b, and μ mRNAs were estimated to be 60-80%, 50-70%, and 50-83%, respectively, on the basis of their hybridization rates with cDNA probes in comparison to mRNA standards of known complexity. Heavy-chain mRNAs of the same class isolated from different mouse strains (Balb/C or NZB) display no detectable sequence differences in cross hybridization experiments, even though the cDNA-mRNA hybrids are submitted to stringent S1 nuclease digestion. These results indicate that allotypic determinants represent only a minor fraction of the heavy-chain constant region sequence in the mouse.
Antibodies were recognized at the turn of the century by their neutralizing effect on injurious microbes or substances foreign to an animal. The ability of the animal to respond to such an invasion in a flexible and specific manner quickly led to imaginative theoretical models to account for this activity. But it was not until the work of Landsteiner that the enormous diversity and exquisite specificity of the antibody system was fully appreciated. For a number of years, the understanding of both diversity and specificity remained a problem suitable only for theoretical speculation, but the birth of protein sequence analysis allowed a fresh look at the antibody problem. It became evident that antibodies were molecules with a defined four-chain basic structure, and in addition, the structural nature of antibody diversity started to unfold with the early comparative amino acid sequence studies of myeloma protein chains. It became clear, therefore, that diversity of antibodies was of genetic origin, and a new wave of theoretical speculation could be put to the test.
A number of fluorescence imaging techniques show diagnostic promise. Imaging endogenous fluorescence has been proposed as a method for cancer diagnosis. Unfortunately, tissue auto fluorescence is relatively weak and poor contrast between malignant and normal tissue is seen. Contrast may be enhanced with the addition of fluorescent materials that are selectively accumulated by malignant cells, such as fluorescein or porphyrin derivatives. The limited penetration of light at the emission maxima of these materials restricts the use of fluorescence techniques utilizing these chromophores to superficial phenomena. However, many potential applications still exist. For example, monitoring fluorescence during surgery may allow resection margins to be clearly delineated.
We have assessed the number of times the gene sequence encoding constant regions of mouse immunoglobulin heavy chains gamma1, gamma2a, and gamma3 are represented in the mouse genome by hybridization kinetic analysis. All three genes are present at one copy each per haploid genome in normal tissues and myelomas producing IgM or IgG3. IgG1-producing myelomas, however, contain 1 copy each of the gamma1 and gamma2a genes and 0.5 copy of the gamma3 gene per haploid genome. IgG2b-producing myelomas contain 1 copy of the gamma2a gene and 0.5 copy each of the gamma1 and gamma3 genes per haploid genome. IgG2a-producing myelomas contain 1 copy of the gamma2a gene and 0.5 copy each of the gamma1 and gamma3 genes per haploid genome. In myelomas producing IgA, all three gamma genes are represented 0.5 times per haploid genome. In order to account for the results we propose an allelic deletion model: (i) The specific deletion of heavy chain constant region genes accompanies the recombination of a variable region gene to a constant region gene. (ii) The portion of the chromosome that resides between two joining sequences is excised out of the chromosome. (iii) The recombination occurs on one of the alleles. Based on this model we also propose that heavy chain genes are arranged on one chromosome in the following order; variable region genes, unknown spacer sequence, mu, gamma3, gamma1, gamma2b, gamma2a, and alpha.
Antibody diversity has a compelling fascination for many scientists and over the years speculations have sometimes seemed more numerous than facts. Now the structural basis of antibody specificity is well defined. Amino acid sequences and recently three-dimensional structures of various immunoglobulins provide the most solid basis for discussing the origin of diversity. The novel pattern of variable (V) and Constant (C) regions of amino acid sequence has been resolved further to show the functional pattern of variability. Inheritance of separate V and C genes is accepted, but attempts to define more than one gene coding for each V region are considered here to be unnecessary. The pattern of variability is still best understood in terms of mutation and the presence or absence of various selective pressures. The major area of debate still hinges around the extent to which mutation and selection operate during evolution or somatically. Sequence data have now been generally interpreted to require multiple V genes carried in the germ line. A few individual VH genes have been mapped in close linkage to CH genes in the mouse. The apparent existence of three VH alleles in rabbits was a strong argument against multiple V genes. Now the three phenotypes have been shown to be due to alleles controlling the expression of three sets of VH genes all present on the same chromosome. That V-gene expression requires rejoining of V and C genes at the DNA level is now almost certain. Models for the joining process can draw on the precedents of transposable genetic elements, which are widespread in Nature. The total extent of antibody diversity remains a philosophical point. Estimates of the number of antibody molecules required for observed diversity are reduced by two recently documented proposals. Each antibody combining site apparently has many (estimated at 100) different specificities and most combinations of VH and VL regions probably form a viable site. A given combining site can be defined by its pattern of shared specificities. Several specific antibody repertoires have been measured and the size in each case is consistent with the stringency with which the specificity is selected. Repertoire size appears to be under genetic control, but there are problems in viewing the genotype through the veil of clonal selection. Molecular hybridization has been used recently in an attempt to count V and C genes directly. C genes are seen in DNA having nonreiterated sequences, as formal genetics predicts. Each V-region probe hybridizes at a similar rate to C-region probes. Interpretation of this result depends on the extent to which one V-region probe will reveal nonhomologous V genes. Previous estimates that many cross-hybridizing genes should have been seen if present are possibly exaggerated. It is argued here that the data are compatible with a germ-line gene for each probe studied. Maximum estimates for the number of germ-line genes are sufficient to account for antibody diversity...
The partially purified immunoglobulin light chain messenger RNA fraction from P3K (MOPC 21) mouse myeloma tissue-culture cells has been employed in hybridisation studies. Fragments of the messenger RNA were generated by alkali hydrolysis. 6-S fragments not containing poly(A) showed the characteristic biphasic hybridisation profile seen with the intact RNA fraction. 12-S and 6-S poly(A)-containing fragments, however, showed single transitions lacking the rapidly hybridising component. Complementary DNA copies of the intact messenger RNA fraction were prepared with RNA-dependent DNA polymerase and the DNA populations fractionated on acrylamide gels. Hybridisation experiments with complementary DNA fractions up to 800 bases in length showed annealing to single (or a few) genes. A rapidly hybridising component (about 200 copies) appears in the cDNA fraction containing the largest transcripts. We conclude that the kappa constant region gene and the MOPC 21 variable region gene are present as one or a few copies in the haploid genome and that the rapidly hybridising component is not due to variable region genes.
We have used purified mouse immunoglobulin light chain mRNA and synthetic DNA which is complementary to it to assess the reiteration
frequency of gene sequences corresponding to the κ constant region of the mouse immunoglobulin light chain. These studies
indicate that the constant region sequence is represented only two to three times per haploid mouse genome, a finding that
rules out a simple stringent germ line mechanism which would require the constant region sequence to be represented hundreds
if not thousands of times. Hybridization studies involving 125I-labeled myeloma light chain mRNA yield interesting results which may eventually permit us to distinguish between the remaining
somatic mutation and recombinational germ line hypotheses. These results reveal a major component of relatively unique frequency
and a minor component with a reiteration frequency of approximately 30 to 50 copies per haploid genome. As discussed, these
results do not permit us to distinguish unambiguously between a germ line model and a type of somatic mutation model that
permits germ line genes corresponding to each κ subgroup. The results do, however, clearly rule out the existence of thousands
of variable region sequences so closely related to the MOPC-41 V-region as to permit extensive stable cross-hybridization.
Die mongolische Heldendichtung zeichnet sich durch die Tatsache aus, daß sie, im Gegensatz zu der bereits literarisch fixierten Heldendichtung vieler anderer Völker, nicht allein auch heute noch lebendig ist und mündlich tradiert wird, sondern daß gegenwärtig noch neue Dichtungen entstehen. Die heldischen Epen der Mongolen bilden eine eigene Provinz innerhalb der Heldendichtung der zentralasiatischen Völker, deren Umfang beträchtlich ist. Allein gegen 150 Aufzeichnungen mündlich tradierter Epen der Burjaten lagern in den Instituten der Sowjetischen Akademie der Wissenschaften. Nur ein Bruchteil davon ist publiziert. Besser erforscht sind die Epen der türkisdien Völkerschaften Zentralasiens, der Kirgisen, Kazaken und der Yakuten. Bei den Völkern Zentralasiens werden von zahlreichen Sängern einige große Zyklen tradiert1, wie das Manas-Epos2 der Kirgisen, das die Odyssee der Kirgisen genannt wird, — dieses Werk umfaßt in seinen rezitierten Versionen 40 000 Verszeilen — ferner das damit in enger Verbindung stehende kirgisische Epos Kökötoy Khan3, das Epos Kyrk Kyz4 der Karal Kapaken zu 20 000 Strophen, die Epen der Kazakhen wie Edüge batyr5 und die usbekische Heldendichtung von Alpamis6. Im Nordosten bei den Yakuten an der Lena wurden Olonkho genannte epische Dichtungen von ebenfalls mehreren Tausend Verszeilen Umfang rezitiert. Allein das yakutische Epos Muldu bogo umfaßt in der Druckwiedergabe mehr als 25 Bogen7.
Recent advances in the technology of nucleic acid biochemistry, in particular the availability of highly purified, light and heavy chain mRNAs from mouse plasmacytomas, have enabled several groups of researchers to study immunoglobulin genes at the molecular level. In this article we will summarize our recent efforts directed to the following two questions:
What is the genetic origin of the enormous diversity of antibody molecules?
How is the genetic information encoded in two seemingly separate segments of DNA, V and C, integrated to generate a contiguous polypeptide chain?
: The nature and extent of antibody diversity are discussed. The genotypic basis of this diversity appears to be multiple V genes and single C genes. A system is described showing coinheritance of the antibody combining site and the V region containing that site. Somatic processes leading to V gene amplification and VC joining are proposed.
RNA preparations containing 70-80% mouse kappa-chain mRNA have been prepared. The remainder consists of many RNA species, each of which represents a small fraction of the total RNA. The kappa-chain mRNA preparation hybridizes with mouse liver DNA with bi-phasic kinetics, indicating that it consists of two fractions -"unique" and "reiterated." Competition hybridization experiments show that the homology among the unique fractions from different mRNAs is the same as the homology among the amino acid sequences of the corresponding kappa-chains. Hence, in addition to the C-region (constant-region) sequences, (most of) the V-region (variable-region) sequences are also derived from unique germ line genes. The reiterated fractions from different kappa-chain mRNAs show essentially complete homology with each other. This fraction seems to consist mostly of sequences which do not code for amino-acid sequences of the secreted polypeptide chain, i.e., the "external" section of the mRNA molecule. It is concluded that the number of germ line genes is too small to account for the observed diversity of antibody molecules.
mRNA coding for the light chain of a myeloma protein has been purified to give one band in acrylamide gel electrophoresis. This pure RNA (S∼13.5) could be translated into the light chain in a heterologous cell-free translation system. The light chain synthesized is apparently slightly larger than the light chain secreted by the tumor.