RESUMEN
During the process of spermatogenesis, the proliferation of spermatogonia (stem cell descendants) is replaced by their differentiation in growing spermatocytes responsible for the preparation to meiosis, which is accompanied by a cardinal change in transcriptional programs. We have demonstrated that, in drosophila, this process is accompanied by a splash of the expression of ß-subunit of nascent polypeptide-associated complex (NAC) associated by ribosomes. Nascent polypeptide-associated complex is known as a chaperone involved in co-translational protein folding. This is the first case of the detection of tissue-specific co-translational NAC cofactor in multicellular eukaryotes. It is proposed that spermatocyte specific NAC is involved in the modulation of the expression of the proteins that provide the functioning of subsequent stages of spermatogenesis.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Chaperonas Moleculares/genética , Espermatocitos/metabolismo , Testículo/metabolismo , Animales , Diferenciación Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Masculino , Meiosis , Chaperonas Moleculares/metabolismo , Biosíntesis de Proteínas , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Espermatocitos/crecimiento & desarrollo , Espermatocitos/ultraestructura , Espermatogénesis/genética , Testículo/crecimiento & desarrolloRESUMEN
The functions of the evolutionary conservative complex NAC (Nascent polypepetide Associated Complex) and its subunits are discussed. The heterodimeric NAC protein contains alpha- and beta-subunits and is found to be reversibly bounded to the ribosome in all eukaryotes, from yeast to humans. NAC contacts the nascent polypeptide and protects it from proteolysis. NAC participates in polypeptide chain folding and modulates protein secretion and transmembrane protein formation. Mutations and deletions of genes, encoding NAC subunits are lethal in early development of multicellular eukaryotes. NAC is involved in the ribosome biogenesis. The beta-subunit interacts with caspase-3 and may be involved in the regulation of the apoptotic pathway. The variants of NAC proteins can be considered as chaperone complexes, involved in the response of the cell and the organism to stress factors, as well as regulators of apoptosis. The genes encoding beta-subunits are rapidly evolved, their duplications cause the formation of tissue specific beta-subunit variants with a different number of putative caspase cleavage sites. The homodimer of alpha-subunits is shown to be the RNA/DNA binding protein and acts as a transcriptional cofactor. The diversity in the functioning of NAC is a prime example of a protein that performs a variety of biological functions (moonlighting protein).
Asunto(s)
Células Eucariotas/metabolismo , Regulación de la Expresión Génica , Chaperonas Moleculares/genética , Biosíntesis de Proteínas , Subunidades de Proteína/genética , Caspasa 3/genética , Caspasa 3/metabolismo , Células Eucariotas/citología , Evolución Molecular , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Unión Proteica , Pliegue de Proteína , Multimerización de Proteína , Estabilidad Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transducción de SeñalRESUMEN
Family of betaNACtes genes in the Drosophila melanogaster genome is a model to investigate the mechanisms of molecular evolution of recently evolved genes. The betaNACtes genes encode proteins, homologous to beta subunit of nascent polypeptide-associated complex (NAC), are expressed in testes and localized on the X chromosome as two two-gene clusters and one separate copy. We collected population polymorphism data for betaNACtes genes using several wild-type stocks of D. melanogaster and compared betaNACtes paralogs with each other. We have shown heterogeneous pattern of betaNACtes genes polymorphism: genes in 3' region of two-gene clusters are low polymorphic, whereas separate betaNACtesl gene is most variable. 5'betaNACtes copies in two-gene tandems are practically identical, whereas 3'betaNACtes copies are highly diverged. Thus, we propose local gene conversion providing selective homogenization of 5'genes. Comparison of betaNACtes paralogs has shown that majority of amino acid differences are in N-terminal part of proteins, containing betaNAC domain. McDonald-Kreitman test of betaNACtes paralog divergence shows the involvement of positive selection in the course of betaNACtes gene family evolution.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila melanogaster/fisiología , Genes de Insecto , Familia de Multigenes , Polimorfismo Genético , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Masculino , Testículo/metabolismo , Cromosoma X/metabolismoRESUMEN
Studies of the position effect resulting from chromosome rearrangements in Drosophila melanogaster have shown that replication distortions in polytene chromosomes correlate with heritable gene silencing in mitotic cells. Earlier studies mostly focused on the effects of euchromatin--heterochromatin rearrangements on replication and silencing of euchromatic regions adjacent to the heterochromatin breakpoint. This review is based on published original data and considers the effect of rearrangements on heterochromatin: heterochromatin blocks that are normally underrepresented or underreplicated in polytene chromosomes are restored. Euchromatin proved to affect heterochromatin, preventing its underreplication. The effect is opposite to the known inactivation effect, which extends from heterochromatin to euchromatin. The trans-action of heterochromatin blocks on replication of heterochromatin placed within euchromatin is discussed. Distortions of heterochromatin replication in polytene chromosomes are considered to be an important characteristic associated with the functional role of the corresponding genome regions.
Asunto(s)
Drosophila melanogaster/fisiología , Eucromatina/fisiología , Heterocromatina/fisiología , Animales , Ensamble y Desensamble de Cromatina , Replicación del ADN , Drosophila melanogaster/genética , Eucromatina/genética , Reordenamiento Génico , Heterocromatina/genética , Cromosoma X/genética , Cromosoma X/fisiologíaRESUMEN
A loss of certain heterochromatic regions (ABO loci) of various chromosomes dramatically distorts the early embryo development in the progeny of females mutant for the abnormal oocyte (abo) gene, which is located in euchromatin of chromosome 2. One ABO locus (X-ABO) is in X-chromosomal heterochromatin distal of the nucleolus organizer. A cluster of the Stellate repeats is located in the same heterochromatin block. Deletions of various fragments from distal heterochromatin were tested for the effect on expression of the abo mutation. The X-ABO locus was assigned to X-chromosomal heterochromatin segment h26 and shown to include repeats consisting mostly of mobile elements and defective Stellate copies. A major part of the regular Stellate tandem repeats proved to be distal of the X-ABO locus.
Asunto(s)
Sistema del Grupo Sanguíneo ABO/genética , Drosophila melanogaster/genética , Heterocromatina/genética , Proteínas de Insectos/genética , Proteínas Quinasas/genética , Secuencias Repetitivas de Ácidos Nucleicos , Cromosoma X , Animales , Southern Blotting , Proteínas de DrosophilaRESUMEN
A full-length copy of the retrotransposon GATE was identified as an insertion in the tandemly repeated, heterochromatic, Stellate genes, which are expressed in the testis of Drosophila melanogaster. Sequencing of this heterochromatic GATE copy revealed that it is closely related to the BEL retrotransposon, a representative of the recently defined BEL-like group of LTR retrotransposons. This copy contains identical LTRs, indicating that the insertion is a recent event. By contrast, the euchromatic part of the D. melanogaster genome contains only profoundly damaged GATE copies or fragments of the transposon. The preferential localization of GATE sequences in heterochromatin was confirmed for the other species in the melanogaster subgroup. The level of GATE expression is dramatically increased in ovaries, but not in testes, of spn-E(1) homozygous flies. We speculate that spn-E is involved in the silencing of GATE via an RNA interference mechanism.
Asunto(s)
Elementos Transponibles de ADN , Drosophila melanogaster/genética , Heterocromatina/metabolismo , Secuencia de Aminoácidos , Animales , ADN/metabolismo , Femenino , Hibridación in Situ , Masculino , Datos de Secuencia Molecular , Mutación , Ovario/metabolismo , Filogenia , Estructura Terciaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Testículo/metabolismoRESUMEN
Molecular evolution and divergence of paralogous tandem heterochromatic repeats Stellate and Su(Ste) located on the X and Y chromosomes, respectively, are discussed. These repeats appear to emerge as a result of amplification of a unique autosomal euchromatic gene encoding the regulatory beta-subunit of the CK2 protein kinase. The autosomal gene and the clusters of heterochromatic repeats are transcribed in testes. A high level of the Stellate expression leads to partial male sterility and disturbed meiosis. The Stellate expression and its adverse effects are suppressed by homologous Su(Ste) repeats. In genome evolution, the open reading frames (ORF) of Stellate and Su(Ste) putative ancestor were maintained by translational selection. Then the coding Su(Ste) function had been damaged and switched to a new one related to antisense Su(Ste) transcription and suppression of the Stellate genes. Symmetrical (sense and antisense) Su(Ste) transcription causes the formation of a double-strand RNA, which by interference (selective switching off the homologous gene expression) suppresses the Stellate genes. The biological significance and the driving forces of evolution of the species-specific balanced interaction of Stellate and Su(Ste) repeats remain mysterious.
Asunto(s)
Drosophila melanogaster/genética , Evolución Molecular , Heterocromatina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Masculino , Datos de Secuencia Molecular , Selección Genética , Supresión Genética , Secuencias Repetidas en Tándem , Cromosoma YRESUMEN
Here we report the peculiarities of molecular evolution and divergence of paralogous heterochromatic clusters of the testis- expressed X-linked Stellate and Y-linked Su(Ste) tandem repeats. It was suggested that Stellate and Su(Ste) clusters affecting male fertility are the amplified derivatives of the unique euchromatic gene betaCK2tes encoding the putative testis-specific beta-subunit of protein kinase CK2. The putative Su(Ste)-like evolutionary intermediate was detected on the Y chromosome as an orphon outside of the Su(Ste) cluster. The orphon shows extensive homology to the Su(Ste) repeat, but contains several Stellate-like diagnostic nucleotide substitutions, as well as a 10-bp insertion and a 3' splice site of the first intron typical of the Stellate unit. The orphon looks like a pseudogene carrying a drastically damaged Su(Ste) open reading frame (ORF). The putative Su(Ste) ORF, as compared with the Stellate one, carries numerous synonymous substitutions leading to the major codon preference. We conclude that Su(Ste) ORFs evolved on the Y chromosome under the pressure of translational selection. Direct sequencing shows that the efficiency of concerted evolution between adjacent repeats is 5-10 times as high in the Stellate heterochromatic cluster on the X chromosome as that in the Y-linked Su(Ste) cluster, judging by the frequencies of nucleotide substitutions and single-nucleotide deletions.
Asunto(s)
Drosophila melanogaster/genética , Evolución Molecular , Heterocromatina/genética , Proteínas de Insectos/genética , Familia de Multigenes , Cromosoma X , Cromosoma Y , Animales , Secuencia de Bases , Mapeo Cromosómico , ADN/genética , Proteínas de Drosophila , Fertilidad/genética , Genes de Insecto , Intrones , Masculino , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteínas Quinasas/genética , Secuencias Repetitivas de Ácidos Nucleicos/genéticaRESUMEN
The X-linked Stellate repeats, encoding a putative regulatory subunit of protein kinase CK2, are expressed in XO male testes. The Y-linked, testes-expressed paralogous Su(Ste) repeats are thought to be suppressors of Stellate transcription. The unique, testis-expressed euchromatic gene was suggested to be an ancestor of the both types of amplified paralogous repeats. A Su(Ste)-like orphon was localized on a Y chromosome, outside of the Su(Ste) cluster. Several diagnostic molecular markers peculiar for the both types of diverged Stellate and Su(Ste) units were detected in the orphon sequence. The orphon was suggested to be a close relative of the immediate ancestor of both types of paralogous repeats which initiated evolution on the Y chromosome. Selection pressure on the level of translation was shown as a driving force in the evolution of Su(Ste) repeats, which are considered as more ancient derivatives of the ancestor euchromatic gene than Stellate repeats. In a vicinity of 12E Stellate cluster the undamaged, recently originated euchromatic Stellate orphon was found at 12D, providing the poly(A) signal for the bendless gene. P-element mediated transformations reveal that the fragments of cloned Stellate and Su(Ste) clusters are able to induce variegation of a reporter mini-white gene. The observed variegation phenomenon has peculiar features: a significant increase of trans-activation of a reporter mini-white gene in homozygous state; absence of effects of several conventional modifiers of position effect variegation (PEV) and independence of a severity of variegation on a distance between insertion and centromere region.
Asunto(s)
Proteínas de Drosophila , Evolución Molecular , Silenciador del Gen , Genes Reporteros , Proteínas de Insectos/genética , Proteínas Quinasas , Proteínas Represoras/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Drosophila melanogaster , Ligamiento Genético , Masculino , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Cromosoma XRESUMEN
The peculiarities of the sequences of 18S rDNA included in a 90-kb DNA segment cloned in YAC vector are described. This heterochromatic segment is situated on the X chromosome distal to the main rDNA cluster. The pseudo 18S rDNA sequence comprised undamaged stretches of rDNA interspersed with segments characterized by high density of nucleotide substitutions and insertions/deletions. The observed patchwork arrangement of unaltered rDNA sequences was considered as evidence of segmented gene conversion events between the normal and damaged genes which are thought to constitute one of the mechanisms of rDNA array homogenization. The 18S rDNA fragment (510 bp) located nearby, homologous to the internal, undamaged part of pseudo 18S rDNA, carries comparable density of randomly distributed nucleotide substitutions with no evidence of correction.
Asunto(s)
ADN Ribosómico/genética , Drosophila melanogaster/genética , Genes de Insecto , Seudogenes , ARN Ribosómico 18S/genética , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Familia de Multigenes , Análisis de Secuencia de ADNRESUMEN
The 30-kb cluster comprising close to 20 copies of tandemly repeated Stellate genes was localized in the distal heterochromatin of the X chromosome. Of 10 sequenced genes, nine contain undamaged open reading frames with extensive similarity to protein kinase CK2 beta-subunit; one gene is interrupted by an insertion. The heterochromatic array of Stellate repeats is divided into three regions by a 4.5-kb DNA segment of unknown origin and a retrotransposon insertion: the A region (approximately 14 Stellate genes), the adjacent B region (approximately three Stellate genes), and the C region (about four Stellate genes). The sequencing of Stellate copies located along the discontinuous cluster revealed a complex pattern of diversification. The lowest level of divergence was detected in nearby Stellate repeats. The marginal copies of the A region, truncated or interrupted by an insertion, escaped homogenization and demonstrated high levels of divergence. Comparison of copies in the B and C regions, which are separated by a retrotransposon insertion, revealed a high level of diversification. These observations suggest that homogenization takes place in the Stellate cluster, but that inserted sequences may impede this process.
Asunto(s)
Drosophila melanogaster/genética , Evolución Molecular , Heterocromatina/genética , Proteínas de Insectos/genética , Familia de Multigenes , Proteínas Quinasas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN , Proteínas de Drosophila , Ligamiento Genético , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos , Cromosoma XRESUMEN
A pseudogene bearing the bulk of the 18S RNA gene was detected outside the rDNA cluster. It comprised irregularly distributed nucleotide substitutions as well as short insertions and deletions. No sequence alterations were observed in the 5' region of the pseudogene, whereas the frequency of substitutions and alterations per nucleotide number in the 3' region and in the middle of the sequence was 7.6% and 1.8%, respectively. The observed sharp irregularity in distribution of substitutions and alterations was considered the result of successive recombinations between the functional 18S rRNA gene and its diverged or damaged variants. This phenomenon provides experimental evidence that recombinations between the pseudogene and functioning repeats of rDNA are implicated in the mechanism of rDNA sequence correction. A segment of the pseudogene sequence was shown to contain substitutions primarily in regions coding for single-strand parts of the RNA molecule. The same segment contained a deletion and an insertion of a nucleotide, approximating it to the most of the studied eukaryotic 18S rRNA sequences. These observations allowed us to supposed that a structural rDNA variant, a fragment of which appears in the pseudogene sequence, is present in the genome. The data obtained suggest both the presence of 18S rDNA variants, and recombination between them, determining the concerted evolution of rRNA genes.
Asunto(s)
ADN Ribosómico/genética , Drosophila melanogaster/genética , Variación Genética , Seudogenes , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Familia de Multigenes , ARN Ribosómico 18S/genética , Recombinación GenéticaAsunto(s)
Clonación Molecular/métodos , ADN , Cromosomas Fúngicos , Genoma Humano , Biblioteca Genómica , Humanos , Transformación GenéticaRESUMEN
A partial genomic library from the Batumi L stock of Drosophila melanogaster was constructed using yeast artificial chromosomes as vectors. The DNA was restricted by Not1 and large fragments were inserted into the YAC5 vector. The size of cloned DNA varied from 90 to 500 kb. 48 random clones were characterized by in situ hybridization to the Batumi L polytene salivary gland chromosome. Single euchromatic sites of hybridization were detected for 27 clones; 11 clones revealed the main euchromatic hybridization site and several additional sites scattered along the chromosomes; 8 clones carried repeats which hybridized to chromocenter and other chromosomal sites; clones with 500 and 90 kb inserts originated from the Y chromosomes and nucleolus, respectively. The library is enriched by the repeated sequences related to the b-heterochromatin.
Asunto(s)
Cromosomas Fúngicos , ADN/genética , Drosophila melanogaster/genética , Genoma , Saccharomyces cerevisiae/genética , Animales , Clonación Molecular , Vectores Genéticos/genética , Hibridación de Ácido Nucleico , Secuencias Repetitivas de Ácidos Nucleicos/genética , Glándulas Salivales/fisiologíaRESUMEN
DNA and RNA specific dyes, Ag-NOR staining and in situ hybridization were used for studying the nucleolar apparatus in the growing oocytes of Henricia hayashi (Asteroidea: Echinasteridae). A plasmid containing ribosomal genes of Drosophila melanogaster (Kolchinsky et al., 1980) labelled with 3H by nick-translation served as an rDNA probe. Multiple extrachromosomal nucleoli are formed by the cascade type as a result of growth and subsequent fragmentation of the chromosomal (primary) rDNA body and its derivative extrachromosomal (secondary) rDNA bodies. Ribosomal genes were shown in all nucleolar structures. Argentophilia of the primary and secondary DNA bodies appears to be due to the dense packing of the rDNA-containing material. Ag(+) NORs were detected in the extrachromosomal multiple nucleoli and NOR complexes. Amplification of rDNA is a highly probable conclusion from the existing data.
Asunto(s)
Nucléolo Celular/ultraestructura , ADN Ribosómico/genética , Amplificación de Genes , Oocitos/ultraestructura , Estrellas de Mar/genética , Animales , ADN/genética , Microscopía Electrónica , Hibridación de Ácido Nucleico , Región Organizadora del Nucléolo/ultraestructuraRESUMEN
In an inbred low-activity (LA) strain of Drosophila melanogaster with a low level of fitness and a complex of inadaptive characters, in situ hybridization reveals an invariant pattern of distribution of three copia-like elements (mdg-1, mdg-3, and copia). Rare, spontaneous, multiple transpositions of mobile elements in the LA strain were shown to be coupled with a drastic increase of fitness. A changed pattern of various types of mobile elements was also observed on selecting the LA strain for higher fitness. High-fitness strains show transpositions of mobile elements to definite chromosomal sites ("hot spots"). Concerted changes in the location of three different mobile elements were found to be coupled with an increase of fitness. The mdg-1 distribution patterns were also examined in two low-fitness strains independently selected from the high-fitness ones. Fitness decrease was accompanied by mdg-1 excision from the hot spots of their location usually detected in the high-fitness strains. The results suggest the existence of a system of adaptive transpositions of mobile elements that takes part in fitness control.
Asunto(s)
Elementos Transponibles de ADN , Drosophila melanogaster/genética , Animales , Selección GenéticaRESUMEN
Poly(A)+ RNA from the lens of the frog Rana temporaria contains three components (1200 +/- 50, 1000 +/- 50, and 900 +/- 50 bp in size) and a more heterogeneous RNA species with a length of 650-750 nucleotides. This RNA was used as a template for the AMV reverse transcriptase and Escherichia coli DNA-polymerase I and the total cDNA obtained was cloned in the PstI site of the pBR322 plasmid vector. Recombinant plasmids corresponding to abundant poly(A)+ RNA classes contain cDNA inserts from less than or equal to 200 to 1200 nucleotides in length. Part of the library (clonotheque) was divided into classes differing in the presence of absence of the restriction sites for BamHI, EcoRI and HindIII restriction endonucleases. The clones belonging to each of the five classes were characterized by the hybridization-translation test. The translation product of mRNA hybridizing with the clone pRT(1)294 has an M4 of about 22 000 and is specifically precipitated by the antiserum to lambda-crystallins of Rana temporaria. The size of the cDNA present in pRT(1)294, equal to 580 +/- 20 bp, is sufficient for coding the greater part of the lambda-crystallin amino acid sequence. On the basis of these data, we conclude that the clone pRT(1)294 codes for one of the frog lambda-crystallins.
Asunto(s)
Cristalinas/genética , ADN Recombinante/análisis , ADN/genética , Genes , Rana temporaria/genética , Animales , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , Escherichia coli/genética , Hibridación de Ácido Nucleico , Biosíntesis de Proteínas , ARN Mensajero/genéticaRESUMEN
Two isozymes of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (G6PD) of Drosophila melanogaster encoded by allelic genes were purified 3000-fold by biospecific phosphocellulose chromatography, using NADP as the enzyme eluent. Electrophoretically fast isozyme A and slow isozyme B variants prove to contain identical subunits with molecular weight of 54000-55000. G6PD is shown to be a dimer. An antiserum directed to highly purified isozyme A does not inhibit the activity of both isozymes. The mutant forms of G6PD restoring the viability of flies without 6-phosphogluconate dehydrogenase show drastically increased Km values for NADP and/or glucose-6-phosphate. It was demonstrated for two mutations that a sharp (200-fold) magnification of Km value for the substrate followed by a considerable increase in the enzyme thermostability might not exert any essential influence on the Km value for NADP.
Asunto(s)
Drosophila melanogaster/enzimología , Glucosafosfato Deshidrogenasa/genética , Isoenzimas/genética , Mutación , Alelos , Animales , Drosophila melanogaster/genética , Glucosafosfato Deshidrogenasa/aislamiento & purificación , Glucosafosfato Deshidrogenasa/metabolismo , Sueros Inmunes , Inmunoensayo , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Cinética , Sustancias Macromoleculares , Peso MolecularRESUMEN
A 1500--2000-fold purification procedure using substrate elution from phosphocellulose is described for two isozymes of 6-phosphogluconate dehydrogenase (6PGD) coded for by the corresponding allelic genes. Taking into account the data of gel filtration and of SDS polyacrylamide gel electrophoresis both isozymes are shown to be dimers containing identical polypeptides of mol. weight 50 000. Antisera against the highly purified sample of 6PGD, inactivated by lyophilization completely inhibited the enzyme activity. Antigens reacting to antisera were revealed by Ouchterlony immunodiffusion tests in extracts of flies carrying the wild type or mutant Pgd allele, coding for 6PGD. In addition to 6PGD antigen (antigen 1) another protein (antigen 2) which shared no common antigenic precipitative determinants with the antigen 1 was revealed in extracts of the normal flies. Antigen 2 was demonstrated also in the six different mutants which expressed zero level of 6PGD activity and had no antigen 1. Mol weight of a 6PGD subunit and of antigen 2 purified by immobilized antibodies were shown to be identical by SDS-polyacrilamide gel electrophoresis. A transformation of "antigen 2" to "antigen 1" was performed by treatment of the former in 2% SDS-mercaptoethanol solution. As a result of SDS treatment no changes of antigenic properties of the inactivated and dissociated 6PGD dimers were observed in immunodiffusion tests.