Illegitimate Gene Transcription or Basal Transcription and its Implication

 

Dr. Yasuhiko Kimoto, Discovery Research Laboratory

Tanabe Seiyaku Co., Ltd., Osaka, Japan

 

 

Abstract

 

The transcription of any gene which occurs in any cell type or tissue is named illegitimate or ectopic transcription. This phenomenon is observed in every single human cell, and is known as basal transcription. This transcription proceeds through usual promoters and probably other usual transcription-related systems. Within a cell, functionally meaningless transcripts and the corresponding proteins are continuously produced as a large amount of waste. Purposeless and constant transcription proceeds as the arrow of time captured in a eukaryotic cell. However, this inevitable chemical reaction, DNA à RNA basal constant flow, could be considered indispensable as a basis of life.

 

Key words: illegitimate transcription, basal transcription, life, chaos, the arrow of time

 

1.      Introduction

Proteins constructing living matter are encoded by genomic DNA and are produced through the translation of messenger RNA (mRNA), which is transcribed from DNA. The amount of protein depends on the amount of transcripts, especially mRNA, and determines the profile of the cells. This fact coincides with the cell differentiation. In the long history of science, studies have usually concentrated on mRNA specifics or characteristics for differentiated cells. The presence of a protein has been discussed whether or not it is necessary and functional for the cell. Transcription of DNA has been considered switched ON when the gene or the protein becomes necessary. The proteins thought to be important for the cell, the so-called housekeeping proteins, are translated from mRNA continuously; the switch for transcription of the corresponding gene is always ON. The story is the same for functionally necessary proteins. On the other hand, as for unnecessary protein, the switch for transcription is believed to be OFF, and the gene is not transcribed.

 

The discovery of illegitimate transcription or ectopic transcription offers a possibility that in cells all transcriptable genes are transcribed into mRNA; all switches for transcription may be always ON. In this article this phenomenon is introduced and it's meaning in life will be discussed.

 

2.      Illegitimate transcription or ectopic transcription

It has been demonstrated that there are many different mRNAs in cells, though relationships between mRNAs and function of the cells is not completely understood. The first report on an ectopic expression of mRNA for a protein with tissue-specific function was published by Humphries et al. (1) and described that mouse globin mRNA existed not only in erythroid cells but also in non-erythroid tissues. When RNA was discovered, two theories were proposed about the transcription of DNA to RNA; one was necessary/unnecessary theory or a switch ON/OFF system, the other was that all genes were transcribed including posttranscriptional regulation. The former has been dominantly supported as a general concept. Later, in 1988 using PCR technique Chelly et al. found human dystrophin gene transcripts in several tissues other than skeletal muscle (2). This phenomenon was confirmed about various tissue-specific gene transcripts for anti-Mullerian hormone, beta-globin, aldolase A and factor VIII in human nonspecific cells such as fibroblasts, lymphoblasts and hepatoma cells (3). Therefore, they estimated that the transcription of any gene occurs in any cell type. This expression of mRNA was named by them to be illegitimate transcription. It was calculated that one copy of mRNA is expressed in 100-1000 cells (4). The start site of this illegitimate transcription was demonstrated to be the same in both specific and non-specific cells; the illegitimate transcription may proceed through the usual promoters (5). Then, they anticipated that all promoters could be minimally active when ubiquitous transcription factors reach their cognate DNA element and gene transcription is probably very low but not zero.

 

Sarkar and Sommer reported on ectopic transcription of blue pigment, factor IX, phenylalanine hydroxylase, tyrosine hydroxylase genes in white blood cells, liver, erythroleukemia cells K562 and chronic villus cells except one example (6).

 

In addition, mRNA for functionally differentiated proteins were detected in normal cells such as peripheral blood lymphocytes, subpopulations of lymphocytes, lymphokine-activated killer cells, sperm, gastric mucosa and 15 established tumor cell lines without any exception using highly sensitive nested PCR method (7). Normal cells of various tissues and tumors share same kinds of mRNA such as steroid hormone receptors, cytokines, lymphocyte surface molecules and both cerebral and peripheral or digestive hormones probably without any relationship to functions. Pregnancy-specific glycoprotein gene transcripts were expressed in male lymphocytes (8, 9). mRNA for male specific protein was also detected in peripheral blood lymphocytes of both sexes (10). Moreover, even non-hematopoietic tumor cell lines express transcripts for heavy-chain constant regions of immunoglobulin gene M through A and of T-cell receptor gene (11). The author anticipated that every mRNA expression occurs in every cell.

 

This phenomenon is not restricted in human beings but probably occurs in all eukaryotes. Ectopic expression of tissue-specific mRNA was detected first in mice (1) and later in rats (12).

 

3.      Illegitimate transcription is a basal transcription

Chelly et al. and Sarkar et al. claimed that this illegitimate or ectopic transcription proceeds in tissues or cell groups; one copy of illegitimate transcript in 100-1000 cells (4), and much less than one copy in a cell (6).

 

On the other hand, total mRNA of a single lymphocyte, a sperm or a tumor cell was investigated by repeated nested PCR concerning 26 kinds of mRNAs, and expression of every mRNA in every single cell was confirmed without any exception (8, 9, 13). Contamination or carry-over, a problem usually accompanied with PCR experiments was strictly managed in these experiments and was negligible because all PCR primers were originally designed and applied only in single cell experiments. The result strongly suggested that all mRNAs exist in any single cell.

 

Taken together, it can be concluded that illegitimate or ectopic transcription occurs in every human cell even though it is a somatic cell or a germ cell sperm or a malignant tumor cell. Every transcript of any transcriptable gene is expressed in every single cell despite the amount of the transcript. A single cell contains not only one set of DNA but also one set of mRNA. This phenomenon should be interpreted as a basal transcription.

 

4. Mechanism of illegitimate/ectopic or basal transcription

Illegitimate transcription was reported to proceed through the usual promoters (5). Various factors such as DNA consensus of promotor, enhancer, silencer and transcription factors are responsible for transcription. These factors are not specific for one gene but common to many genes. Therefore, transcription is determined by the net balance of quality and quantity of transcription-related factors. Probably such molecules exist simultaneously in a cell, and transcription can be initiated whenever elementary molecules react each other. As Chelly et al. anticipated, the illegitimate or basal transcription never becomes null. The basal transcription could be interpreted as DNA à RNA basal constant flow (7).

 

Transcription factors have been eagerly studied recently to reveal the mechanism of transcription. These transcription factors construct a complex of several functionally different molecules. For example, TFIIH consists or RNA polymerase II, CDK-activating kinase (cdk7 and cyclin H), DNA helicase and other subunits (14). Cell division, transcription and DNA repair proceed almost simultaneously. Production of RNA or mRNA can occur during serial chemical reactions of cell duplication and cell division.

 

5.      Immune system and illegitimate or basal transcription

This illegitimate or ectopic transcription can elucidate the immunotolerance against autologous antigens. The majority of the autologous antigens synthesized and expressed stochastically on thymus cells are presented during any stage of development (15). However, any gene transcripts and translated proteins are expressed not only each thymus cells but also other somatic cell and even immune cell itself. Clonal elimination or deletion and clonal anergy could take place both in the thymus and in the peripheral organs.

 

Recently isolated malignant cell-specific or dominant proteins such as MAGE (malignant melanoma-associated protein) or MUC1 (breast cancer-associated mucin antigen) are considered being produced and expressed also in the peripheral blood lymphocytes because mRNAs for these proteins were detected in lymphocytes (9). Patients’ autologous cytotoxic T lymphocytes can be induced in vivo and in vitro and recognize these autologous antigen molecules means that these proteins may be biochemically modified and can be recognized as non-self. However, the majority of the proteins preserve the authentic structure even in tumor cells. Therefore, there may exist autoimmune system in normal body, and recognition of self proteins depends on the balance of the amounts of recognized molecules and recognizing molecules. This hypothesis can account for clonal anergy.

 

 

6.      Detection of genetical disorder by using mRNA

The fact that all mRNAs exist in every cell makes it possible to detect the abnormality of genes in hereditary diseases using mRNA substitute for genomic DNA. Eluted DNA from abnormal affected tissues has usually been analyzed. However, some tissues such as neurons or deep organs are not accessible. Instead of these, organs easily prepared cells such as peripheral blood lymphocytes are available. Moreover, it is reasonable to analyze mRNA encodes the sequence of amino acids of proteins including exon variant or DNA rearrangement.

 

In this decade following Chelly et al. abnormalities in genes have been detected in patients’ peripheral blood lymphocytes, fibroblasts or Epstein-Barr virus-transformed lymphoblastoid cell lines. In addition to abnormal sequences like deletion or point mutation, such abnormalities as exon variants, abnormal or alternative splicing or duplication can only be detected by investigating gene transcripts. Patients or carriers of Duchenn and Becker muscular dystrophy were diagnosed by analysis of dystrophin mRNA, and gene rearrangements, point mutations, deletion, duplication and exon variants were revealed to produce abnormal dystrophin proteins (16-22). Point mutations in factor VIII gene of hemophilia A patients and duplication in factor IX of hemophilia B patients were also indicated using peripheral blood lymphocytes (23-25). Loss of exons caused by mutations in introns also occurred in peripheral blood lymphocytes in addition to alternative splicing, mutations and deletion of bases in exons (26-29). Such abnormalities were also detected as illegitimate transcription in persistent Mullerian duct syndrome (30), familial hypertrophic cardiomyopathy (31), spondyloepiphyseal dysplasia (32), hypoparathyroidism (33), x-linked Alport syndrome (34), phenylketonuria (35, 36), inherited osteoarthritis (37), cystinuria (38), and Glanzmann thrombasthenia (39).

 

For the purpose to reveal relationship between disease and abnormalities of not only genomic DNA but also mRNA and translated proteins, mRNA should be applied for investigation.

 

7.      Detection of micrometastases

It is a critical point for prognosis of malignant diseases whether there are metastases of malignant cells circulating in blood vessels. When mRNA for dominantly expressed protein in malignant cells can be detected in patients’ blood or lymph nodes, micrometastasis is strongly suspected (40-43). However, the more sensitive the method for detecting mRNA is, the more possible the illegitimate or basal transcription expressed in normal cells is detected (44, 45).

 

There is no method to distinguish mRNA coding for one protein in malignant cells from that in normal cells. Only tumor specific abnormality in genes is distinguishable. Therefore, a method like highly sensitive PCR cannot be recommended to detect normal mRNA for the diagnosis of micrometastasis. In order to achieve this purpose the amount of aimed mRNA expression in a single cell, a normal cell or a malignant cell, must be determined (46-48).

 

8.      Possibility of dedifferentiation and redifferentiation

In every eukaryotic cell whole mechanism of transcription works, every transcriptable DNA is transcribed and every mRNA is expressed. If relative concentration of molecules responsible for transcription of one mRNA changed, the amount of the corresponding mRNA would change. Then, it may be possible to change the course of differentiation. Differentiated cells could be dedifferentiated under other conditions and could be induced towards another differentiation.

 

Transplantation of a nucleus of G₀-arrested udder cell to an enucleated oocyte developed a viable lamb (49, 50). One of the critical points to make a cloned lamb was that the mature differentiated mammary cell should be arrested in G₀ stage by serum starvation. It could be estimated that the starved cells nearly reached the undifferentiated condition only with the basal transcription. After fusion of the nucleus with enucleated oocyte its cytoplasm could offer different concentrations of transcription-related molecules that were enough for the cell containing G₀-arrested nucleus and the basal transcription to be incubated as a fertilized egg.

 

In forthcoming era of tissue engineering, human embryonic stem cells will be available for the treatment of damaged tissues and organs. The functional cells induced and differentiated from embryonic stem cells, however, have a problem concerning rejection of transplantation caused by antigen mismatch. If we discover a technology which can change the differentiated cells to be dedifferentiated and a technology which can induce them to any differentiated functional cells necessary for medical treatment, we can use our own cells or tissues.

 

 

9.      The arrow of time in a cell

The illegitimate or basal transcription could be considered as a serial chemical reaction that produces RNA from DNA without any purpose along with time: the arrow of time (51) captured in a cell. This chemical response is fundamental for life and naturally occurs according to chemical inevitability when molecules necessary for the reactions are present. As time passes, purposeless basal transcription proceeds to one way, which is determined by an initial condition such as relative amount of chemical substances. After it reaches one dynamically constant state it will develop into another stable condition through a crisis point. This deterministic chaos may make living matter with enormous profiles and differentiations.

 

Some biologists found fractal organization controlling structures all through the body. Branching bronchus or the vessels proved fractal (52). The cells constructing these bronchus and vessels may also fractal. This concept can be included in the story of Chaology, a new science which accounts for nature through mathematics, physics and chemistry.

 

Duve mentioned in his theory of life that the origin of life, evolution of life and development of large number of living matter must be controlled by the same process and the same rules as those in non-living (53, 54). The collection of the inevitable chemical reaction constructs a cell. It could be negligible that biological process initiates with some purpose.

 

A large part of chemical substances present or produced in a cell is probably junk, and possesses no functional meaning for the cell at lower concentration. There still remains another problem: whether all mRNAs are translated to proteins in the cytoplasm. Is there a posttranscriptional regulatory system to determine the amount of the proteins? Some mRNA, for example beta-actin, is present in large amounts in every cell. Even though a posttranscriptional regulatory system exists, the quantity of the most proteins is considered to depend dominantly on the amount of corresponding mRNA. Translation occurs when mRNA are transported to the ribosomes. The quality of an mRNA to survive in the cytoplasm is thought to be same by itself in different cells. It is not mRNA itself but circumstances that determine the rate and the amount of mRNA and the translation to proteins. Like the basal transcription mechanisms of the basal translation work as time passes.

 

Regardless, in order to function for the maintenance of life, a considerable number of the molecules is necessary. From this functional point of view, living matter undergoes a tremendous waste to consume energy for producing unnecessary or functionally meaningless substances.

 

Among the collection of possible chemical reactions, only some creatures fit for the condition and the circumstances of the earth can survive (survival of the fittest). As a result of the inevitability of chemical reactions, illegitimate or basal transcription happens, which can elucidate the possibility and the polymorphism of living matter. The phenomenon of illegitimate transcription or basal transcription means that the presence of living matter is identical with the possibility of purposeless, inevitable chemical reactions.

 

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Dr. Yasuhiko Kimoto received his Medical Degree from the Osaka University School of Medicine, and began his career at the Department of Surgical Oncology at the Research Institute for Microbial Diseases at Osaka University as a surgeon. He continued his medical research while enhancing his education with the pursuit of a Ph.D., which he received in 1988. Dr. Kimoto is presently General Manager of the Discovery and Research Laboratory at Japan's Tanabe Seiyaku Co. Ltd. (Editor's note: for a brief history of DNA/RNA research, please refer to: The First Steps in the USA and in France Toward Great Discoveries In RNA and DNA in this issue of the Journal).