JPH04197180A - Matrix protein gene of human parainfluenza 2 virus and substance containing the gene - Google Patents

Abstract

NEW MATERIAL:The subject human parainfluenza 2 gene containing the unit expressed by the total or a part of the formula as the total or a part of the base-sequence constituting the gene. USE:Diagnostic agent and therapeutic agent for PIV-2 infection disease, etc. PREPARATION:The objective gene can be synthesized by phosphoamidite process based on the base sequence having complementarity to the gene RNA of PIV-2.M protein of formula.

Description

[Detailed Description of the Invention] Field of Application of Soil] The present invention relates to a gene that coats the matrix protein (hereinafter referred to as M protein) of human parainfluenza virus type 2 virus (hereinafter referred to as PI V-2). In more detail, the P IV-2 M protein gene RN
Since it shows complementarity to A and roughly contains the entire coding region of the PIV-2 M protein, it can be used as a gene useful as a diagnostic agent or therapeutic agent for PIV-2 infection, for example.

[Conventional technology and its problems]

For example, in order to enhance the therapeutic effect of viral infections, it is necessary to identify the infectious virus and perform appropriate treatment based on the identification results.

Conventionally, viruses have been identified based on their serological properties, and the main methods are enzyme-linked immunosorbent assay (hereinafter referred to as ELISA), neutralization reaction method, and complement fixation reaction. method, hemagglutination inhibition reaction method, fluorescent antibody method, agar precipitation reaction method, etc. are known. However, in both of these methods, the determination is made by measuring antibodies against the virus in the specimen, and in order to make a highly accurate determination, - The virus antibody titer increases one week after infection during boat fishing. This has to be done at the beginning of the disease, and in some cases, it may be necessary to take a re-measurement several weeks later for confirmation.Therefore, there is a problem in that it is difficult to make pre-symptomatic and early diagnosis.

Usually, the ELISA method is used to measure PIV-20), and this method measures antibodies against PIV-2 in a specimen. For example, a specimen is placed on a plate immobilized with PIV-2. After adding and reacting, washing, adding anti-PIV-2 antibody to a colander and reacting in the same manner, then adding an enzyme-labeled antibody, reacting, and developing color, this method is also the same as above. It is difficult to make pre-symptomatic and early diagnosis, which inevitably delays the start of treatment, making it difficult to increase the therapeutic effect.

The present invention makes it possible to solve the above-mentioned problems, and particularly provides a gene useful for a test agent that enables early diagnosis of PIV-2g infection.

[Means to solve the problem]

The present invention provides that all or a part of the base sequence constituting the gene has the following [formula]% TCATTGGGTCACATCCCCAA'GACT
GCACTTCCTTTTTTCAGCAAGATG
GGATTGCCTGTCATCCATTACAAGA
TGTTTCCCCTAATCTAGCAAATCA
CTGTGGTCAGTTGGATGTGAGATAA
GATCTGCCAAGTTGATACTTCAAGA
ATCTGATCTTAATGAGCTAATGGGGC
CACCAGGACCTTATATCACTGATAAGA
TTGCCATTAGATCAGGTCAACGGAC
ATTTGAGAGGTCCAATTCAGCCCAT
TCAAAATATGCATCAATTCCAAAACT
TGGAAGCCATCAACTGAATGCTCCA
GCATCTGAGAATAGAACCACAATCA
AGTCACTACTACTAGTCACTATACAA
TAATCAACAATTTTAGTCAACTGAT
TACCAAGATGTTATCATAGGTCCGA
A gene (hereinafter referred to as a DNA
(sometimes abbreviated as fragments),
This is an attempt to solve the problem of difficulty in pre-onset and early diagnosis of PIV-2 infection.

The DNA fragment provided by the present invention is PIV-2.
Since it shows complementarity to the gene RNA of M protein, it is called PI.
When used as a test agent for V-2 identification, it is possible to directly identify PIV-2, and it is possible to solve problems in the conventional closed identification method using antibody measurement. Since the -2.M protein gene contains the entire coat region of the PIV-2.M protein, the expressed M protein can be used as an antigen when producing antibodies or as a raw material for vaccines. It is also useful as a therapeutic agent for PIV-2 infection.

The method for producing the DNA fragment represented by the above [formula] in the present invention is not particularly limited and can be carried out in accordance with a conventional method.For example, (a) mRNA obtained from PIV-2g stained cells
The cDNA thus prepared is introduced into an appropriate vector such as an E. coli plasmid vector and cloned into E. coli. 1. A method of screening using nucleocapsid RNA as a probe; 2. A method of screening using nucleocapsid RNA as a probe; 2. A method for screening using oligonucleotides synthesized by PIV-2, 2. A method for screening using an antibody against PIV-2, etc. PIV-2・M! A method of isolating a clone encoding the white gene and separating insert DNA from the plasmid of the isolated clone, (b) Complementary to the gene RNA of the PIV-2-M protein represented by the above [formula] The phosphoramidite method (N
ature, Vol. 310, p. 105, 1984), (c) a method of combining the above (a) and (b), and the like.

The DNA fragment of the present invention obtained by the above method [is
It can be used as follows.

For example, the DNA fragment obtained by the above method is further cut into ten or more bases, preferably twenty or more bases, with an appropriate restriction enzyme (e.g., BglII, etc.) if necessary, and then labeled with a radioactive isotope or the like for inspection. After reacting this test agent with a nylon membrane, etc. on which RNA extracted from a specimen (pharyngeal fluid, etc.) is immobilized, the test agent is tested using the Northan hybridization method (hereinafter referred to as the Northan method), etc., which is evaluated using autoradiography, etc. Identification of PIV-2 can be performed.

In the present invention, the unit represented by all or a part of the above [formula] is useful as a test drug, etc., but the unit can also be appropriately incorporated into an NA vector and used in the same manner as above. Furthermore, the vector does not necessarily have to be a DNA vector, and may be incorporated into or mixed with other appropriate substances such as polymers, cellulose, glass beads, pharmaceuticals, etc. Can be used.

[Examples] The present invention will be described in more detail below based on Examples.

Vero cells were grown in Eagle's essential medium containing 5% fetal bovine serum, and then this culture was injected into Eagle's essential culture containing freshly prepared actinomycin D (2 μg/ml). Next, PIV-2 (Toshiba stock) was inoculated and cultured for 24 hours in the same culture solution.After detaching the virus-infected cultured cells with a trypsin/EDTA mixture, they were incubated at 8000 rpm for 10 hours. Cells were collected by centrifugation for 1 min and treated with guanidine isothiocyanate solution (6 M guanidine isothiocyanate, 5 m M sodium citrate, 0.1 M 2-mercaptoethanol, 0.5%
Sodium lauroyl sarcosinate) was added, quickly dissolved in a homogenizer, and the chromosomal DNA was sheared by passing it through a syringe barrel equipped with a 21G needle 5 times. The obtained cell lysate was centrifuged again at 8000 rpm, and 9 ml of the obtained supernatant was layered in another centrifuge tube containing 3 ml of a 5.7 M cesium chloride aqueous solution, and the mixture was placed in a Beckman rotor (Beckman 5W40Ti rotor).
Ultracentrifugation was performed at 18°C for 18 hours at 37,000 fpm, and the RNA pellet was collected after centrifugation.

From the RNA obtained as above, oligo dT cellulose Type 7 (Pharmacia
a)) was used to extract mRNA containing poly A chain (hereinafter referred to as p
o ly (A+) RNA] was isolated and purified.

2 cDNA The La Poulie cDNA library was synthesized according to the Okayama-Barb method. That is, 5 μl of distilled water was added to 4 μg of the above poly(A+) RNA, heated at 65°C for 10 minutes, and then rapidly cooled.
Oligo dT was added to the Kpn I site of the vector (
0.8-1.2 μg/μl)] 10 μl, synthesis reaction solution (
500mM Tris-HCl buffer pl (8,3,300
mM potassium chloride, 80mM magnesium chloride, 3mM
dithiothreitol) 3μl and 20mM dATP
, 20mM dCTP, 20mM dGTP, 20m
Add 1 μl of MdTTP to each and add 20 uni
After adding ts RNasin and 40 unjts reverse transcriptase, the total volume was made up to 30 μl with distilled water, and reaction was performed at 42° C. for 30 minutes to synthesize second-strand cDNA. After completion of the second strand cDNA synthesis, 10 to 30 dG bases were added using terminal deoxynucleotidral transferase in the presence of dGTP. Next, the DNA was digested with restriction enzyme I (indm), annealed with a linker having a dC base chain, and then closed into a circle with E. coli ligase.Finally, in the presence of RNase, the DNA was
Second strand synthesis was performed using polymerase to create complete plasmid DNA. Competent cells (DHI) obtained by subsequent calcium chloride treatment 100~
10 μl of a mixed solution of 0.4 M magnesium chloride, 10.1 M calcium chloride, and 0.02 Mg of the above plasmid DNA were added to 200 μl, and the mixture was incubated at 0°C for 40 minutes, and then incubated at 0°C for 40 minutes.
℃ for 90 seconds, then culture solution (Bactodributon 1
0g, yeast extract 5g, sodium chloride 5g
1.5ml of a solution of g dissolved in 10100O distilled water)
was added and left at 37°C for 40 minutes to obtain transformed cells.

By introducing 0.02 Mg of plasmid DNA into 200 μm competent cells (DHl) as described above, 2500 independent clones were obtained in the presence of ampicillin.

3 Leo PARNA Rho no-,'P I
0.6% nonidetsu) P-40 in V-2 transfected Vero kidney cells
, a solution containing 10mM vanadyl ribonucleotide complex (0.15M sodium chloride, 0.05M Tri
s-hydrochloric acid buffer) was added, solubilization was performed by vipetating in water for 1 hour, and centrifugation was performed at 8000 rpm for 10 minutes. Next, add 1 ml, 2.5 ml, and 1 ml of a 40% aqueous solution, a 30% aqueous solution, and a 25% aqueous solution of cesium chloride, respectively.
In the centrifuge tube layered in this order, 9 ml of the supernatant obtained from the above centrifugation was further layered, and the tube was placed in a Peckman rotor 5.
Equilibrium density gradient centrifugation was performed on W40Ti at 37,000 rpm for 18 hours at 16°C. After centrifugation, the virus nucleocapsid band generated in the 30% cesium chloride aqueous solution layer was collected. Next, 0.1% SDS and proteinase K (2.5 mg/m 1 ) were added, and the mixture was heated at 56°C for 15 min.
After proteolysis for a minute, phenol and phenol/
Nucleocapsid RN was treated with a chloroform mixture.
I got an A. 50 m to the obtained nucleocapsid RNA
Add MTris-hydrochloric acid buffer pH 9.7 and incubate at 95°C for 1
After heating for 0 minutes, the RNA solution was gradually cooled to room temperature. Next, 20 μm of this RNA solution was added with a buffer solution (250 mM Tris = hydrochloric acid, 50 mM magnesium chloride, 25 mM D
T, 7.5 mM spermine, 500 mM potassium chloride), 10 μl, and 32PATP (100 μCi/6
00pM) 3μl, T4-DNA force-inase 2uni
After adding ts, the total volume was adjusted to 50 μm with distilled water and heated at 37°C.
The mixture was reacted for 1 hour to obtain a nucleocapsid RNA probe.

4. Colony Hardization 100 to 200 μl of the above transformed cells were added with ampicillin (120 μg).
Add 15 cm agar plate (10 g bactodributon, 5 g yeast extract, 5 g sodium chloride, 15 g agar) containing 10 g bactodributon, 15 g agar, and bring the total volume to 1000 g with distilled water.
ml) and cultured at 37°C for 12 hours.

Copy the colonies that have formed on the nitrocellulose membrane, and
The cells were incubated at ℃ for 6 hours. The membrane was then treated with a 0.5N sodium hydroxide/1.5M sodium chloride mixture for 10 minutes, followed by a 0.5M solution containing 1.5M sodium chloride.
Tris-hydrochloric acid buffer pl (8°C) for 10 minutes, rinsed in a mixture of 0.3M sodium chloride and 10.03M sodium citrate for 5 minutes, and after rinsing, the membrane was air-dried at 80°C. The DNA was fixed on the membrane by vacuuming for 1 hour and then baking.

For each 5 membranes obtained in this way, denatured salmon RNA
(1mg/ml) 1mL 5SPE solution (3°6M sodium chloride, 200mM monosodium phosphate, 20m
MEDTA) 7.5ml, Denhard solution (2%
A hybridization solution consisting of 1.25 ml of BSA, 2% Ficoll, 2% polyvinyl and lolidon and 31.25 ml of 10% 5D was added and allowed to react at 42°C for 12 hours, and then added to the freshly prepared prehybridization solution. Nucleocapsid RNA probe,
It was added to give a concentration of 5 million Ci/ml, and the reaction was carried out at 42°C for 18 hours.

After the reaction, 20-fold diluted 5S containing 0.1% SDS was added.
After washing twice in PE solution at 42℃ for 5 minutes, 0.1% SD
155 at 42°C in a 200-fold diluted 5SPE solution containing S.
A two-branch wash was performed. After washing, the membrane was air-dried and exposed at -80°C using an X-ray film (RX5, manufactured by Kodak).
Autoradiography was performed for 2 hours and several positive clones were obtained.

5-Zan The Northern method was performed using the above positive clones. That is, poly (A+) RNA derived from PIV-2 infected cells obtained by the method (1) above, poly (A+) RNA derived from virus-uninfected cells, and rR.
After electrophoresing each NA marker in a 1.5% agarose gel, they were transferred to a nylon membrane (Hybond-N, Amajam Co., Ltd.), air-dried, and then irradiated with UV to separate RNA.
was covalently bonded to a nylon membrane, and then hybridization was performed using each of the above positive clones as probes.

That is, the nylon membrane was reacted in the above prehybridization solution at 42°C for 12 hours, and then 100% of the 32p-labeled probe made from each positive clone was incubated.
It was added to the hybridization solution at a concentration of 10,000 Ci/ml and reacted at 42°C for 18 hours. After the reaction was completed, washing was performed in the same manner as in (4) above, and autoradiography was performed using X-ray film.
One clone hybridizing to the se position was obtained.

This clone was named 9M2M.

In addition, the probe of each positive clone is
After digestion with ind m and EcoRI, the insert DNA was separated by electrophoresis using low melting point agarose.
After extraction, it was obtained by random prime labeling using 32PdCTP.

6 PIV-2-M ″-
The clone pM2M obtained in step (5) above was digested with various restriction enzymes to create a restriction enzyme map, and the results are shown in FIG.

That is, FIG. 1 shows that clone pM2M was
After cutting with the restriction enzymes EcoRI, Pvun, and Pvun, the resulting fragments were introduced into the multiple cloning site of plasmid pUC118, and SEQUENA
Sea fencing was performed using the SETM kit (manufactured by Toyobo Co., Ltd.), and some Delasi Yong mutants were created using the Kilo Sea Fence Deresion Kit (manufactured by Takara Shuzo Co., Ltd.), sea fencing was performed, and the base sequence was determined. In the drawing, 1 and 3 are vector DNA, and 2 is insert DNA.
A is the DNA fragment of the present invention.

The results were as shown in formula c above.

7 When using the entire west (7-1) insert DNA of PIV-2: The 9M2M clone obtained in (5) above was cut with Sma I and Sac I, and then electrophoresed using low melting point agarose. A band migrating at a position of about 1400 base pairs was cut out, extracted, and the insert DNA was recovered by ethanol precipitation.

Obtained insert DNAI u l (10pm.

les/μl) per buffer solution (0.5M Tri
s-HCl pH 7, 6, 0, 1MMgC12, 50mM dithiothreitol, 1mM spermidine hydrochloride, 1mM
EDTA) 2μL Distilled water 11.4μL.

(r32P)ATP5a 1 (2Mmo I e
s/lt J), bacteriophage T4 polynucleotide kinase 8 units, then incubated at 37°C for 4 hours.
It reacted for 5 minutes. Next, the reaction solution was subjected to Bioge IP
60 (Japan Bio-Rad Laboratories, Inc.) was used as a probe.

Next, RNA derived from PIV-2g-stained cells obtained by the method (1) above and RNA derived from virus-uninfected cells were mixed at 0.3
After plotting 5 Mg each on a nylon membrane, air drying, UV irradiation, and 4
The reaction was carried out at 2°C for 12 hours. Next, the tip of the probe was placed in a boiling water bath for 2 minutes and then rapidly cooled to 1,000,000 Ci/
ml, and the mixture was further reacted at 42°C for 18 hours. After the reaction was completed, it was washed twice for 5 minutes at 42°C in a 20-fold diluted 5SPE solution containing 0.1% SDS, and then washed with 0.1% SDS for 5 minutes.
Washing was performed twice for 15 minutes at 42° C. in a 200-fold diluted 5SPE solution containing % SDS. After cleaning, air dry the membrane and
12 hour leap autoradiography was performed at -80°C using line film.

As a result, in the area where RNA derived from PIV-2 infected cells was plotted, a positive signal generated by the same probe that had formed a hybrid with PIV-2 viral RNA in the sample was observed, indicating that the cells were infected with PIV-2. This was confirmed. In contrast, RN from virus-uninfected cells
No reaction was observed in the area where A was plotted.

(7-2) Inser) When using a partial DNA fragment The 9M2M clone obtained in (5) above was
After cutting with D, the band that migrates at a position of about 800 base pairs is cut out by electrophoresis using low melting point agarose, and D
After extracting the NA fragments, they were recovered by ethanol precipitation. The obtained DNA fragment was used as a probe by random prime labeling.

That is, 10 μm of distilled water was added to 0.1 Mg of DNA fragment, and the mixture was cooled rapidly by placing in boiling water for 2 minutes. Next, 1 μl of bovine serum albumin (10 mg/m 1 ), random solution (0
44M HEPES pH6,6,100mMTri
s-HCl pH 8, 11mM MgC12, 22mM 2-mercaptoethanol, 44MM dATP, 44MM
dGTP, 44MMdTTP, 0.12mMTri
s-salt wL pH 7,5,0,12mM EDTA, 1l
units/ml oligonucleotide pd(N)a (Pharmacia stock % formula % n1ts) was added and allowed to react at room temperature for 6 hours. The resulting reaction solution was purified using Bio-ge 1P60 and used as a probe.

Next, 0.35μ of RNA derived from PIV-2 infected cells obtained by the method (1) above and RNA derived from virus-uninfected cells was added.
After plotting each g on a nylon membrane, air-drying, UV irradiation, and incubating in the hybridization solution of (4) above for 42 hours.
The reaction was carried out at ℃ for 12 hours.

Next, the probe was placed in a boiling water bath for 2 minutes, then quenched and then added to a concentration of 1,000,000 Ci/ml, followed by further reaction at 42° C. for 18 hours.

After the reaction is complete, add 20-fold diluted 5-fold diluted solution containing 0.1% SDS.
Washed twice for 5 min at 42°C in SPE solution, then 0.1%
1 at 42°C in 200-fold diluted 5SPE solution containing SDS.
External washing was performed for 5 minutes. After washing, the membrane was air-dried, and autoradiography was performed at -80°C for 12 hours using X-ray film.

As a result, in the area where RNA derived from PIV-2 infected cells was plotted, a positive signal generated by the same probe that had hybridized with PIV-2 viral RNA in the sample was observed, indicating that the cells were infected with PIV-2. This was confirmed. In contrast, RN from virus-uninfected cells
No reaction was observed in the area where A was plotted.

(7-3) When using an oligonucleotide synthesized based on the base sequence represented by the above formula c; the above [formula]
Based on the base sequence shown in , an oligonucleotide of ten or more bases can be synthesized and used as a probe.

DNA synthesizer (DNA synthesizer, model 381A
(Manufactured by Abride Biosystems Japan Co., Ltd.)
Oligonucleotide synthesized using (5'-TAT
GCTCGAGACACTTCAG-3') was purified using an oligonucleotide purification cartridge (Applied Biosystems Japan Co., Ltd. 11). The resulting synthetic oligonucleotide I B l (10 pmo
per l e s/# l), 2 μl of the buffer solution (7-1) above, distilled water 11°4 μm, (732P)ATP 5 μl
After adding 8 units of bacteriophage T4 polynucleotide F kinase (2 nmoles/μl) and reacting at 37° C. for 45 minutes. Next, the reaction solution was
-gelP60, and the obtained purified product was used as a probe.

Next, RNA derived from PIV-2g-stained cells obtained by the method (1) above and RNA derived from virus-uninfected cells were mixed at 0.3
After plotting 5 μg each on a nylon membrane, it was air-dried, UV irradiated, and reacted in the hybridization solution (4) above at 42° C. for 12 hours.

Next, the above probe was added at a concentration of 1,000,000 Ci/ml, and the reaction was further carried out at 42°C for 18 hours.

After completion of the reaction, 5-fold dilution containing -0.1% SDS was performed.
Washed twice for 5 min at 42°C in SPE solution, then 0.1%
1 at 42°C in 200-fold diluted 5SPE solution containing SO5.
External washing was performed for 5 minutes. After washing, the membrane was air-dried, and autoradiography was performed at -80°C for 12 hours using X-ray film.

As a result, in the area where RNA derived from PIV-2 infected cells was plotted, a positive signal generated by the same probe that had hybridized with the PIV-2 virus RNA in the sample was observed, indicating that the cells were infected with PIV-2. It was confirmed that On the other hand, no reaction was observed in the area where RNA derived from virus-uninfected cells was plotted.

When expressing a protein encoded by the 9M2M clone; 9M2M clone contains almost the entire region of the M protein gene and M
Since it contains the entire gene region that encodes the protein, it is possible to express the protein encoded in the gene by inserting the inserter DNA of the 9M2M clone into an appropriate vector. The expressed protein can be used as an antigen for producing antibodies, as a raw material for vaccines, etc. Various methods can be used for protein expression, for example, in vitro expression using rabbit red blood cell lysate, etc.
In vitro synthesis systems, systems using microorganisms such as Escherichia coli and Bacillus subtilis, systems using cells such as yeast, insects, and mammals can be used to express proteins encoded by various genes.

The start codon in the insert DNA of the 9M2M clone (
ATG, the above-mentioned [bases 80 to 82 shown in the formula] are 1 from IO counting from the ribosome binding site of the expression vector pKK 223-3 (manufactured by Pharmacia Co., Ltd.).
Make sure it is at the 5 base pair position. That is, a deletion insert DNA was prepared by removing the base sequence from number 0 to number 79 shown in the above [formula], and pKK 223
-3 vector was introduced into the Sma1 site.

The obtained clone was introduced into E. coli JM109, and YT culture solution containing 100 μg/ml of ampicillin (16 g of Bactodributon, 1 Yeast Extract
Dissolve 0g of sodium chloride in distilled water to make 11) and incubate at 37°C for 12 hours. Next, add isopropylthiogalactoside to 2mM and
Further culture with shaking is performed at ℃ for 5 hours. After the culture is completed, the culture solution is centrifuged at 11,000 Orp for 10 minutes at 15°C to obtain a precipitate. Next, the precipitate was washed with 30mM Tris-HCl buffer (pH 7.5) containing 30mM sodium chloride, suspended in the above buffer, and 1 mg of lysozyme and 25 μl of 0.25M EDTA were added per 1ml of suspension for 15 minutes. After standing at room temperature, freeze-thaw four times. Next, this melted liquid was heated at 11,000 Orp for 60 minutes at 4°C.
Centrifuge to obtain the supernatant. The target protein in the obtained supernatant was identified using the method of Komada et al. (J, gen, V
iroh 1989, Volume 70, No. 3487~
3492 pages) and the method of Nageshige et al. (Archivesof
Virology, 1987, Volume 95,
Pages 211-224) etc. That is, anti-PIV-
After immunoprecipitation using 2 antibodies, analysis was performed by polyacrylamide gel electrophoresis, and it was confirmed that M protein was expressed in E. coli.

〔Effect of the invention〕

The gene represented by the above formula provided by the present invention is specifically complementary to the gene RNA of PIV-2 M protein, and therefore can be used as a test agent for early diagnosis of PIV-2 infection. Furthermore, since the gene represented by the above formula contains the entire coding region of the PIV-2 M protein, the expressed M protein can be used as an antigen for producing antibodies or as a raw material for vaccines, etc. Therefore, it is extremely useful in that it can be applied to both the inspection and treatment of PIV-2 infection at the same time.

[Brief explanation of the drawing]

FIG. 1 is a restriction enzyme map of the cDNA region of the DNA (9M2M) that is complementary to the P IV-2 gene represented by the above formula. 1 and 3...Vector DNA, 2...◆...
insert DNA.

Claims (2)

[Claims] (1) All or part of the base sequence constituting the gene is
A human parainfluenza type 2 virus matrix protein gene which is a unit represented by all or part of the following [formula]. [There is a gene sequence] [There is a gene sequence] (2) All or part of the base sequence constituting the gene is
A substance containing the human parainfluenza type 2 virus matrix protein gene, which is a unit represented by all or part of the following [formula]. [There is a gene sequence]