Tissue-specific regulation of the expression of ceruloplasmin (Cp) gene, which encodes major copper-containing extracellular glycoprotein was investigated. A decrease of the Cp concentration associated with copper amounts in milk during the first 3 days of lactation was used as phenotypic index for evaluating the Cp enzyme activity in the mammary gland. Computer analysis of mammalian Cp gene promoter region has revealed conserved sequences of cis-elements, which potentially were capable of regulating the enzyme activity. It has been shown that changes in the nucleotide sequence of specific transcriptional factor binding sites located at 5′-end of Cp gene were associated with disturbance of the regular downregulation of Cp gene activity during lactation.

A study was made of the expression of the ceruloplasmin (Cp) gene, whose product, blue multicopper ferroxidase, acts as a neuron survival factor. Computer analysis predicted an alternative promoter in the 3′-terminal region of intron 2 of the rat Cp gene and showed that transcription from this promoter potentially yields an mRNA coding for a 109-kDa polypeptide. The alternative Cp form starts with a region of 25 amino acid residues encoded by a part of intron 2. From Gly113, its sequence is identical to that of the major Cp form. The in silico data were confirmed experimentally by RT-PCR. The predicted mRNA was found predominantly in the liver and brain of adult rats. Direct sequencing of the PCR product demonstrated the complete identity of the predicted and real mRNA sequences. It was shown in vitro that a Cp-like protein of about 110 kDa is synthesized in the cytosol and accumulates in the absence of mitochondria. The protein was transferred into isolated mitochondria in a reconstructed system. Transport was energy-dependent, was associated with no change in Cp polypeptide length, and required cytosolic factors. Mitochondrial import of the Cp form was probably determined by an internal mitochondrial localization signal, KVVYREFTDSTFRE, corresponding to region 756–769 of mature Cp. The possible role of Cp in mitochondrial iron metabolism is discussed.

Alternative expression of ceruloplasmin (Cp) gene, whose product, blue multicopper ferroxidase, is a neuron survival factor, was studied in the current work. Computer analysis showed that Cp-mRNA isoform, coding for 109 kDa polypeptide, can be formed as a result of the transcription from the alternative promoter in 3'-region of intron 2 of rat Cp gene. Alternative Cp form starts with 25 amino acid residues sequence, coded with intron 2 region. It is followed by amino acid sequence of the main Cp isoform starting from Gly 113. In silico data were experimentally confirmed using RT-PCR. It was demonstrated that the predicted mRNA was generally localized in liver and brain cells of adult rats. Direct sequencing of the obtained PCR-product showed the entire coincidence of the real and predicted mRNAs. It was in vitro showed that approximately 110 kDa Cp-like protein was completed and accumulated in the absence of mitochondria. This protein is transferred into the isolated mitochondria in the reconstructed system. Transport is energy-dependent, it is not accompanied with the shortening of Cp polypeptide length and needs the presence of cytosolic factors. Probably import is determined by the inner protein mitochondria import signal with amino acid sequence KVVYREFTDSTFRE, located in 756-769 region of mature Cp. Possible role of Cp in iron metabolism in mitochondria is under discussion.

A site of rat DNA (about 1800 b. p.) adjacent to the first ceruloplasmin gene contains, apart from regulatory sequences common for all eukaryotic promotors, cis-elements, which are potential binding sites for soluble nuclear receptors of some hormones. Sequences characteristic of genes expressed in liver cells and mammary gland cells during lactation were detected. Full-length fragment of this locus of ceruloplasmin gene (1800 b. p.) was synthesized by PCR and used in gel shift experiments. It was found that soluble proteins extracted from purified nuclei of mammary gland cells during lactation and from the liver of adult and newborn rats, contain proteins specifically interacting with the PCR product. A fragment of chromosome gene containing exons encoding the central part of rat ceruloplasmin was cloned in pTZ19 bacterial vector. Gel shift assay showed that the cloned fragment contained binding sites for specific transcription factor YY1, whose level in nuclear protein fractions varied during ontogeny (according to immunoblotting data). Monoclonal antibodies detected protein YY1 in the complex of cloned DNA-nuclear proteins. Possible mechanisms of tissue-specific regulation of ceruloplasmin gene varying during ontogeny are discussed.

Primate genomes contain a very large number of short interspersed GC-rich repeats of the Alu family, which are abundant in introns and intergenic spacers but also present in 5′ flanking regions of genes enriched in binding motifs (BMs) for transcription factors and frequently containing CpG islands. Here we studied whether CpG islands located in promoters of human genes overlap with Alu repeats and with clusters of BMs for the zinc-finger transcription factors Sp1, estrogen receptor α, and YY1. The presence of estrogen-response elements in Alu was shown earlier and here we confirm the presence in the consensus Alu sequence of the binding sites for Sp1 and YY1. Analyzing >5000 promoters from the two databases we found that Alu sequences are underrepresented in promoters compared to introns and that ∼4% of CpG islands located within the −1000 to +200 segments of human promoters overlap with Alu repeats. Although this fraction was found to be lower for proximal segments of promoters (−500 to +100), our results indicate that a significant number (>1000) of all human genes may be controlled by Alu-associated CpG islands. Analysis of clustering of potential BMs for the indicated transcription factors within some promoters also suggests that the Alu family contributed to the evolution of transcription cis-regulatory modules in the human genome. It is important that among Alu sequences overlapping with CpG islands in promoters a large fraction of members of the old Alu subfamilies is found, suggesting extensive retroposon-assisted regulatory genome evolution during the divergence of the primates.

By screening with labeled Alu DNA, a clone was isolated from cDNA expression library, which appeared identical in sequence to the well-known Ca-phospholipid-binding protein annexin II. To evidence the DNA-binding activity of recombinant annexin II and its presence in the cell nucleus, we have expressed full-length mouse annexin II cDNA in bacteria with pGEMEX vector. The expressed protein was studied with electrophoretic mobility shift assay and for its reaction with polyclonal antibody to chromatin-associated ribonucleoprotein (alpha-RNP), which is one of the major acid-dissolvent components of the nucleus. The obtained results confirm the DNA-binding activity of recombinant annexin II. Annexin II reacts with polyclonal antibody to rat alpha-RNP. So, annexin II is a major nuclear DNA-binding protein in mammalian cells.