The easy automation of DNA sequencing has greatly facilitated in the characterization of mammary cDNA and genes associated with milk proteins in various species. Some 60 cDNA and 20 genes from 12 species have already been completely sequenced (Bonsing et al., 1988). These cDNA or genes can now be modified in vitro by site directed mutagenesis and then expressed in various systems, such as bacteria, yeast, Bculovirus infected cells and Cos cells after insertion into adequate vectors. Studies of genetic polymorphism at the nucleotide level have led to the discovery of new alleles (Roberts et al., 1992). Expression analysis of native or modified genes using in vitro transcription systems, mammary cell lines, and transgenic animals has greatly improved the knowledge of the functioning of milk protein and has offered the potential for the genetic modification of milk composition (Persuy, 1992).
Over the last years, using biotechnology, researchers developed mice, which demonstrated promoter elements with tissue specificity driving on hormonally regulated fusion DNA to express products, (Reddy, 1991) however, only a limited number of promoter elements have been introduced in to domestic farm animals.
Materials and Methods
A high molecular weight DNA was isolated from lactating goat mammary gland by using methods described by Sambrook et al., 1989. It was not an easy task to get very pure goat genome DNA and the required length of the fragment. By using phenol and ether, highly pure Genome was extracted, which was between 23 and 19kb long. First Enzymatic Digestion Pilot Experiment was performed to establish the exact ratio of enzymes to DNA required to fully digest the DNA then Sau3 was used to digest the DNA. In order to isolate the required length of fragments from the rest, the Sucrose Gradient Centrifugation technique was applied (Sambrook et al., 1989) and analyzed by electrophoresis by using λ HindIll Marker as indicative of size.
All the tubes which contained fragments 23<19kb long were combined and used for further construction of the Genome library. Before constructing the library it was also necessary to purify the fragments because recovered fragments by Sucrose Gradient centrifugation contained sucrose. Elutiploid and 2X low salt and high salt buffer were used to purify the fragments. After the elution was completed the DNA fragments were precipitated with 2 volume of ethanol and recovered by centrifugation at 12000 g for 10 minutes and dissolved in 50 μl TE pH 8.00 and were stored in -20°C. At this stage purified fragments were ready to be legated into a vector λ arm. The bacteriophage EMBL3 was the phage used to prepare pure λ DNA. The EMBL3 phage was selected because it can accommodate large fragment of genome DNA, up to 20 Kb, and because this vector is particularly useful for cloning sau3 partial digests (Sambrook et al., 1989) and also because BamHl sites in the vector are flanked by Eco RI and Sall sites and, therefore, it was possible to release the cloned fragment by digesting the recombinants with Sail and EcoRl. It was not ideal to clone the fragment into the plasmid vector as the fragment was 23 Kb <19Kb, therefore, the plasmid vectors could not accommodate it. To get a vector, which can accommodate such large fragments, isolation and purification of λ DNA from EMBL3 phage was performed following the method of Sambrook et al. (1989) and digested with Bam HI and the results of the digestion mixture, were analyzed through electrophoresis. The Right and left arms were recovered from the gel and purified through phenol and chloroform extraction. (Sambrook et al., 1989). When the arms were ready they were legated to the genome fragments in order to achieve a probe specific to a promoter of β-casein gene, primer one and primer two were designed (Roberts et al., 1992) in the up stream from TATA Box. Primer I consisted of 20 nucleotides (ATG, CTC.CCC, AGA, ATT, TCT, GO) and primer II 20 nucleotides (TAT.ATA, CGA, TAT, TCA, ATG, AC) also. Polymerase chain reaction products were recovered and through λ marker the length of the product was investigated as size indicative marker. The PCR product was not enough to be used directly as probe therefore, it was needed to amplify the probe in PUC 18-plasmid vector. The 135bp long was amplified in plasmid vector and then the vector was linearized by digesting it with single enzyme without releasing the 135bp long probe and directly used to screen the goat genome library constructed, PCR was used for screening the library of the goat genome DNA, (Sambrook et al., 1989) so that the unneeded recombinant phage was reduced.
The 4.5 kb long .5-casein promoter was cloned into plasmid vector and amplified. (Sambrook et al., 1989). The template DNA for sequencing reaction was purified by standard method. The DNA used in the sequencing reaction, as template was not single strand, rather double stranded and was denatured by alkali in an annealing mixture consisting of annealing buffer, and forward and reverse primers. Then the annealed reaction was labeled using T7 DNA polymerizes labeling buffer and a 32PdATP. 135bp nucleotides from 5' to 3' and 110 bp nucleotides from 3' to 5' flanking of the 4.5 kb long promoter were sequenced.
High molecular weight of goat genome has been isolated from goat mammary gland. For the results of the electrophoresis (Fig. 1) the Goat Genome DNA isolated from Goat Mammary Gland is highly pure. The first lane on the left is lambda Hindlll. The goat genome was digested partially with sau3A. After sucrose gradient centrifugation at 22,000 rpm for 22 hours at 20°C, the desired size was pooled for construction of the genome library in bacteriophage λ EMBL3. The partial digests of the goat genome fragments analyzed through electrophoresis (Fig. 2). Goat Genome fragments were of different size due to Sau3A partial digest. The first lane on the left is lambda Hindlll. Polymerase chain reaction products with very small molecular weight on the right (Fig. 3). On the left is the Goat Genome used as control and in the middle is lambda Hindlll marker. The PCR fragment was cloned into Puc18 vector (Fig. 4) which was used as probe to screen the library. The λ DNA was isolate from EMBL3 Phage (Fig. 5), highly concentrated lambda DNA isolated from EmbL3 phage, and digested with BamHl. The left and right arms (Fig. 6), in the middle is the staffer of the phage lane and fifth from right to the left is Hindlll marker. The arms were isolated from the gel, ligated to Sau3A fragments and packaged. The Genome Library was then constructed and screened by in-situ hybridization. The appearance of black dots indicates recombinant phage colonies (Fig. 7). The colonies harboring the recombinant phage are big and separate and their black colour is very prominent. Sa generated fragments of the recombinant phage were clone in pGEM5Z and were sequenced (Fig. 8). The 4.5 kb fragment were cloned into pGEM5z.The second lane from left is lambda Hindlll marker .The first lane from the left is pGEMSz harboring the 4.5kb fragment. The result of the two directional sequence of the gene shows partial sequence of the 4.5kb of the Goat beta casein Regulator) Elements (Fig. 9).
|Fig. 1:||High molecular weight Genome Goat DNA.
1- From left A HindIII Marker.
2-7 Genome Goat DNA
|Fig. 2:||Sau3A fragments of the goat Genome.
1-From left A Hind III marker.
2-13 Goat Genome fragments.
A goat genome library was constructed in the EM vector using the DNA fragments of 23<19kb long. Approximately a genome library consisting 2.1X105 were screened. Five positive clones were obtained and analyzed by restriction enzymes and polymerase reaction (Sambrook et al., 1989). The DNA was sequence and sequence data were analyzed by reading the results the autoradiograph film (Sambrook et al., 1989).
|Fig. 3:||Results of PCR using the goat Genome as template.
1-From left the Goat Genome as control
2-From left λ HindIII marker.
3-From left PCR product.
|Fig. 4:||PCR fragment cloned in Puc18
|Fig. 5:||A DNA isolated from EmbL3 phage
Lanes 1-4 from right cntain, BamHl fragments. Each lane ontains three fragments one of which is in the middle, the stuffer and the other tow fragments are the arms of the phage. The fifth lane from right contains lambDA Hindlll marker
|Fig. 7:||Results of Hybridization, the black dots indicate recombinant phage colonies
The target DNA to be sequenced was 4.5kb long. 135bp from 5' to 3' flanking region of the 4.5kb DNA fragment inserted into plasmid vector was sequenced and 110bp from 3' to 5' flanking was sequenced. Due to the importance of this promoter it is suggested that a further research using this promoter to drive foreign gene in the culture of mammary gland epithelial cells and finally in the mammary gland of a goat or other domestic animals.
Isolation of high molecular weight goat genome from goat mammary gland was performed following procedure described by Ebert et al. (1989). Vector phage consisting left and Right arms of Lambda EmBL3 phage were purified and packaged with packagene.
|Fig. 8:||1- From right Hindlll marker
2- From right the cloned Gene into pGEMS 5z
|Fig. 9:||Results of sequence of the isolated regulatory elements of β-casein Gene.
Goat Genome Library consisting 2.1x105 was screened by using in-situ hybridization and polimerase chain reaction (PCR). Fragment of 4.5kb was cloned in PGEMZ5 and was partially sequenced. Expression of active transgene prodcuts extraneous to the target tissue may seriously affect the physiology of the animal (Behringer et al., 1989). Therefore, more restrictive promoter elements are necessary to be cloned from milk protein genes. The 4.5kb long fragment of milk protein gene regulatory element isolated may restrictively drive the expression of any gene in the mammary gland.
Some discrepancies occurred with very low expression it transgenic mice for rat β-Casein transgen e comprising a 3.5kb5 flanking region and 3kb3 flanking region (Lee et al., 1988), In contrast, expression was high by stage specific an mammary tissue specific in transgenic mice carrying Cprine Beta-Casein transgene with 3kb5 flanking region a 6kb3 flanking region (Persuy, 1992).
Transgenic experiments have demostrated that why protein genes possess more compact promoter and gi better expression than the casein with the exception goat Beta-Casein (Persuy, 1992).
The findings of this study are in accordance with the findings of Roberts et al. (1992). Using these Regulatory Elements to drive the expression of foreign gene in the mamm gland, unlike other regulialory elements may specific expression only in the mammary gland of large and small domestic animals.