Cysteine 50 of the POU H domain determines the range of targets recognized by POU proteins.

The best target of POU proteins (Oct-1, Oct-2) is an octamer sequence ATGCAAAT. POU proteins also recognize, with weaker affinity, the TAAT-like targets of another group of regulatory factors, the homeoproteins. Up to now, it has not been known why Cys50 of the POUHdomain is absolutely conserved in contrast to that in homeoproteins. To assess the importance of Cys50 in determining the binding specificity of POU proteins, all possible amino acids were substituted for Cys at position 50, and the resulting mutants were tested with probes containing octamer (ATGCAAATNN) or homeospecific binding sites. Only the wild-type POU was shown to adequately discriminate between the octamer and homeospecific sites, and the protein affinity was only slightly affected by the nucleotide sequence flanking the octamer at the 3'-end. Any amino acid substitution at position 50 resulted in the mutant protein binding efficiently both to the octamer and the TAAT-like sequences. Moreover, in this case the 3'-flanking sequences influenced the binding to a much greater extent.

Conservative Val47 residue of POU homeodomain: role in DNA recognition.

Conservative Val47 residue, located in the third recognition helix of the Oct-2 POU domain, was alternately substituted with other 19 amino acids. Affinity and specificity of interaction with oct-site ATGCAAANGA and homeo-specific site ATAANGA were determined for all mutants. The wild type protein (with Val47) has maximal affinity and specificity in POU domain interaction with octamer sequence. However, V47I mutant showed stronger interaction with homeo-specific site. The highest specificity of interaction with homeo-site was recorded for V47S mutant. We conclude that only Val47 provides sequence-specific high-affinity binding of POU proteins with octamer targets other than the homeo-specific site. It is shown also that damages caused by point mutations may be at least partially compensated by participation in the oct-site recognition of both POUh and POUs domains.

[Proteins, interacting with the promotor of the immunoglobulin kappa gene]. / Belki, vzaimodeistvuiushchie s promotorom kappa-gena immunoglobulina.

Two octanucleotide binding protein factors (mOct-1 and mOct-2A) are detected in nuclear extracts of lymphoid NS/0 cell line. These proteins, corresponding to human Oct-1 and Oct-2A nuclear factors, were purified by ion exchange and heparin-agarose chromatography. With retardation assay technique it was shown that mOct-2A factor at all steps of purification contains tightly bound protein, dissociating in the presence of a nonspecific native DNA. This octa-protein-associated factor (OAF) has low affinity to DNA and is not able to bind specifically the promoter of kappa-gene. However it interacts with DNA together with mOct-2A in highly specific manner but does not form complexes with mOct-1 and mOct-2B factors. Since OAF can be attached specifically only to mOct-2A (which play an essential role in initiation of kappa-gene transcription) this attachment may be considered as necessary for its recognition as a specific transcription factor. The ability of OAF like alpha TIF factor (specifically interacting with Oct-1) to discriminate between the Oct proteins, recognizing the same cis-acting oct-elements, shows how the non-DNA-binding transcription factors can influence differentially the transcription by two proteins binding to the same DNA sequences.

[The relative content of oocyte and somatic 5S rRNA at different stages of embryonic development as an index of the replacement of maternal ribosomes by ribosomes of the embryo in the loach]. / Otnositel'noe soderzhanie ootsitnoi i somaticheskoi 5S-rRNK na raznykh stadiiakh émbrional'nogo razvitiia kak pokazatel' zameny materinskikh ribosom ribosomami zarodysha u v'iuna.

Relative content of the oocyte and somatic 5S rRNA in loach Misgurnus fossilis L. during development was determined electrophoretically. Embryos before hatching contain 70% and swimming larvae no less than 50% of the oocyte 5S rRNA. We assume that the relative content of 5S rRNA fractions reflects the proportion between ribosomes synthesized during oogenesis and those synthesized in embryos and larvae. We calculated using previous data (Timofeeva, Kafiani, 1964) the rates of maternal ribosome decay and ribosome synthesis in the embryo. During organogenesis these rates appear to be 1.17-1.09 x 10(6) and 1.7 x 10(6) molecules/sec per embryo, respectively.

[Immunoglobulin genes in lymphoid cells and regulation of their transcription]. / Geny immunoglobulinov v limfoidnykh kletkakh i reguliatsiia ikh transkriptsii.

The hybridoma genomes contain polyploid sets of immunoglobulin genes. We have shown, that the hybridoma PTF-02 genome contains three genes of heavy chains and two genes of light chains. The genes responsible for antibody synthesis were cloned and their structure were determined. Investigation of the kappa gene transcription and its fragments which contain regulatory sequences revealed a nuclear factor. The latter interacts with the octanucleotide localized at the promoter region of the kappa gene. The purified factor activates the transcription of the kappa gene in a heterologous cell-free system. Together with the tissue-specific factor there is also an universal factor interacting with the octanucleotide sequence. We have shown an additional factor in lymphoid cells interact with the protein which binds to the octanucleotide sequence. We have shown an additional factor in lymphoid cells interacting with the protein which binds to the octanucleotide sequence. As a result, there is a family of factors which interact with ATTTGCAT sequence. One major factor (m.w. 60 +/- 2 kDa) is an obligatory component for the initiation of immunoglobulin genes transcription.

[Protein factors, interacting with the octamer sequence of immunoglobulin genes]. / Belkovye faktory,, vzaimodeistvuiushchie s oktaposledovatel'nost'iu immunoglobulinovykh genov.

Nuclei of different cell lines contain protein factors interacting with octamer ATTTGCAT. Fragment k53 of kappa-gene promoter region was used as DNA-probe. The factors from lymphoid cells yield a DNA/protein complex with mobility B0. The proteins are referred to as HF-B0. The nonspecific ubiquitous factor present in many non-lymphoid cells (for instance, HeLa cells) interacts with the probe to produce a complex whose mobility is much lower. The protein NF-B0 was isolated from the nuclear extract of myeloma MOPC21 cells. It was purified by chromatography on ion exchangers, hydroxylapatite, heparin-Sepharose and affinity sorbent containing a synthetic octamer sequence. At all the steps of purification, protein fractions were chosen for their ability to interact selectively with the octamer yielding a complex with the mobility B0. As a result, NF-B0 protein (60 +/- 2)kDa was purified 6.10(4) times to the electrophoretically homogeneous state. Purified factor NF-B0 selectively interacts with the octamer.

[Factors interacting with promoter region of immunoglobulin genes. Determination of the binding site boundaries]. / Faktory, vzaimodeistvuiushchie s promotornoi oblast'iu immunoglobulinovykh genov. Ustanovlenie granits uchastka sviazyvaniia.

The nuclear extracts of plasmacytomas producing antibodies were found to contain factors which formed complexes with the promoter fragment of the gene for immunoglobulin kappa-chains. The corresponding complexes found in the extracts of nonlymphoid cells had a different mobility. Two approaches were proposed for determining the boundaries of the region necessary for protein factors to be bound to DNA using nuclease Ba131. A 5'-ATTTGCAT-3' octanucleotide sequence was shown to be necessary for interaction with the protein nuclear factor in the studied plasmacytoma lines. The protein completely lost its affinity if at least one nucleotide was removed or substituted at the 5'- or 3'-end of this sequence. The procedures proposed for determining the precise boundaries of the sequence necessary for protein binding to DNA do not require a preliminary protein purification. The principles on which the procedures are based, set no limitations to their application to other systems used for studying the interaction of proteins with DNA.

[Factors of tissue-specific transcription of immunoglobulin kappa genes]. / Faktory tkanespetsificheskoi transkriptsii kappa-genov immunoglobulinov.

Proteins TIF1 and TIF2 with mol mass 90 and 94 kD, respectively, were found in myeloma cells. They bind to the promoter region of the kappa genes. Protein TIF1 was found in nonlymphoid cells (HeLa, liver cells). From myeloma MPOC21 a fraction was isolated enriched 400 fold with TIF2 by fractionation on ion-exchangers DE52 and P11 and hydroxylapatite. The purified protein protected the DNA site that contained 30bp including the conservative sequence TNATTTGCAT located upstream of the kappa gene promoter against the action of DNAase. Addition of the purified factor to the extract from HeLa cells increased several fold the efficiency of transcription and corrected the initiation of transcription.