Identification of growth hormone gene duplication in local

Growth hormone or somatotropin is a peptide hormone released by anterior pituitary, it is a small protein that contains about 191 amino acids in a single chain with two disulphide bridges between cysteines at positions approximately 53 and 165 and between 182 and 189. GH and has a molecular weight of about 22,000. The primary structure of GH has been worked out from about fifty species. It causes growth of almost all tissues of body that are capable of growth. Not surprisingly there is a considerable variation in the structure of GH from one species to another, if GH of the same species is injected in any organism, it causes increased growth of that organism but if injected with a GH from a different species it causes slight or no growth effect as it has a different structure and is attacked by immune system of recipient organism. Production of larger quantities of GH is a remarkable achievement of biotechnology, causing increased milk and meat production. GH is synthesized as prehormone with an extension of 25 amino acids. The hormone contains four antiparallel a-helices arranged in a left twisted helical bundle. Aside from its effect on linear growth GH increases rate of protein synthesis, increases mobilization of fatty acids and decreases rate of glucose utilization. Several variants of GH exist in many higher animals due to growth hormone gene duplication, differential mRNA splicing and differential GH protein possessing. Growth hormone causes its effects on target cells by binding to its receptor; a single growth hormone molecule binds with extra cellular domain of two receptor molecules. Some of the growths promoting functions of GH are also mediated by somatomedins, the insulin like growth factors.

2.2 Gene duplication

Gene duplication is a phenomenon in which more then one copies of a segment of DNA are formed in the genome. Gene duplication can be of several types. These are usually classified according to the extent of the genomic region involved. The following types of duplication are recognized: (1) partial or internal gene duplication, (2) complete gene duplication, (3) partial chromosomal duplication, (4) complete chromosomal duplication, and (5) polyploidy, or genome duplication. The growth hormone gene duplication is complete gene duplication. Gene duplication has its evolutionary significance and an unnecessary duplicate of a gene may acquire divergent mutations as at least now one gene is available to perform physiological functions and its duplicate is free from the and evolutionary pressure to save the conserved sequences so duplicate gene emerge as a new gene with some different exon sequences and a greatly differing intron sequences. The greatly differing intron sequences are due to the fact that introns are totally free from evolutionary pressure whereas exons have a pressure as they have to code for a functional polypeptide. The principal molecular mechanism responsible for gene duplication is unequal crossing over. Unequal crossing over between misaligned sequences gives rise to a tandemly duplicated region on one chromosome and a complementary deletion on the other. Once tandem duplication arise in any individual, then by inbreeding it is possible that some descendents will be duplication homozygotes carrying a total of four copies of duplicated gene. Similarly heterozygotes would have three copies and non-duplication homozygotes would have two. Bridges (1936) observed gene duplication for the first time, in the Bar locus of Drosophila. The a and b chains of hemoglobin are an example of duplicate genes. In some instances gene duplication can increase the transcription of any particular type of proteins, as multiple temples are available for transcription, for example silk worm has a tandem array of more than 200 structural genes coding for a egg shell protein, which is required in larger amounts. But GH gene duplication has no such effect on growth parameters, as explained later. Gene duplication should not be confused with gene amplification, as duplication is a process in which any gene forms many copies permanently and gene copy number remains the same in all physiological conditions and is transferred to next generation. While gene amplification is increase in gene copy number in somatic cells to fulfill the increased transcriptional need under some physiological stress and is not transferred to next generation.

2.3 Growth hormone gene duplication

The growth hormone gene is about 1800 bp with four introns and five exons in mammals. In case of some fish GH gene have five introns and six exons. In most mammals a single gene codes for pituitary growth hormone and is not associated with closely related genes. An exception occurs in higher primates; in man a cluster of five very similar genes located over a distance of 50,000 bp on chromosome 17 codes for GH like proteins. One of these on 5¢ end of the cluster; hGH-N codes for pituitary GH. While the other four codes for genes expressed in placenta, including two genes hCS-A and hCS-B for chorionic somatomammatrophin one gene hGH-V for GH variant and one hCS like gene. In rhesus monkey five genes codes for GH like proteins, one expressed in pituitary and four in placenta. There is also evidence of multiple GH like genes in a new world monkey.

Until recently it appeared that duplication of GH gene in mammals is confined to primates. Placental lactogens are found in some other mammalian groups (ruminants, rodents) but appear to have arisen independently of those found in primates, by the duplication of prolactin gene. However a number of reports have now appeared indicating that there are duplicate GH genes in at least some caprine ruminants (goat and sheep). In case of sheep two alleles of GH are found; one allele Gh1 is represented by only one growth hormone gene, while in second allele Gh2 two growth hormone genes are found (GH2-N and GH2-Z) as shown in figure. A similar condition is found in goat. In sheep three genotypic possibilities are Gh1/Gh1 where gene copy number is two or Gh1/Gh2 with gene copy number three or Gh2/Gh2 where gene copy number is four.

Intron Exon

Gh 1

GH 1

Gh 2

GH 2 N GH 2 Z

Fig 1: Organization of growth hormone genes in a sheep of genotype Gh1/Gh2 the segment of DNA shown hare is about 10 Kbp. The arrows indicate the duplicated regions

2.4 Pakistani breeds of fat tailed sheep

Fat tailed sheep are so named because they can store large amounts of fat in the tail and the region of the rump. There is only one species of sheep ovis aries and about 250 different breeds, about 25% of breeds are fat tailed. In Pakistan several fat tailed breeds are found, mostly in NWFP. Some commonly known breeds are The Balkhi is a fat tailed mutton type. It is found in the NWF Province of Pakistan and tribal areas body color varying from black, tan, gray or their admixture the wool yield is 2.0 kg. The ears are moderately long, the body is muscular and compact, and a tucked up fat tail. The Baluchi originated in the area that is now southwest Pakistan, eastern Iran and southern Afghanistan. The wool is coarse with modulation. Body size varies between 35 and 40 kg in adult ewes, milk yield between 40 and 50 Kg in a period of about 125 days. The Hasht Nagri is a fat tailed mutton and wool type. They are found in the Hasht Nagar tract in NWFP. They have a compact body with short legs. The hanging fat tail may reach fetlock. In well-fed animals, the Fat tail may even touch the ground. The Lati is a fat tailed mutton and wool breed found in the Salt Range hills and the surrounding areas including districts of Rawalpini, Attock and Jhelum and parts of Mianwali and Sargodha districts in Punjab the tail is a medium sized hanging fat tail.

2.5 Evolutionary significance

The studies on GH gene duplication have its evolutionary significance. The duplicates arisen as a result of duplication of a single GH gene found in most mammals and other tetra pods. It seems likely that duplication occurred during the evolution of caprine ruminants, after divergence of these from other bovid groups. The polymorphism seen in sheep and goat must therefore also have arisen before the divergence of these two species and have been sustained through speciation and during the following 5–7 million years. In mammals the structure of pituitary growth hormone (GH) is generally strongly conserved, reflecting a slow basal rate of molecular evolution. However, on a few occasions the rate has increased-markedly during the evolution of primates and artiodactyls, and to a small extent during the evolution of rodents and rabbit.

2.6 Effect of GH gene duplication on growth

As far as effect of growth hormone gene duplication, on growth of sheep is concerned, there is no significant effect of GH genotype on any parameter of growth or body composition. It is also shown that GH copy number has no effect on the growth hormone mRNA production. The sequence studies of sheep placental GH (probably the product of the GH2-Z gene) shows an other interesting effect of GH gene duplication. The sequences and molecular modeling techniques showed that placental duplicate of GH has two important substitutions in receptor binding sites, i.e. Gly®Arg at position 9 and Gly®Ser at position 63. These two positions are in two binding sites of GH. The substitution at position 9 leads to enhanced affinity at second binding site where substitution at position 63 causes a carbohydrate group introduction and hence no binding at first binding site, so there is a possibility that this placental duplicate can act as an inhibitor of pituitary GH.

3. Review of literature

3.1 Duplication of GH gene in caprine ruminants (sheep and goat)

Ø On the basis of studies on a restriction fragment length polymorphism (RFLP), it had been concluded that there are two alleles at the GH gene locus in sheep and goats. In sheep, in one allele (Gh1), the GH gene was represented by a single copy (GH1 gene), while in the other (Gh2) the GH gene was duplicated (GH2-N (5¢) and GH2-Z (3¢) genes). Restriction maps of the sheep Gh1 and Gh2 loci indicated that the GH1, GH2-N and GH2-Z genes were all very similar. The sequences flanking the 5¢ ends of the GH1 and GH2-N genes were similar, but differ from that flanking the GH2-Z gene. Individual animals were homozygous for Gh1 (i.e. possessed 2 GH-like genes) or Gh2 (4 GH-like genes) or heterozygous, with one copy of Gh1 and one of Gh2 (3 GH-like genes). The frequency of the Gh2 allele was greater than that of the Gh1 allele. (Valinsky et al. 1990)

Ø It was shown that the GH2-Z gene contained a PvuII polymorphism in the second intron (Gootwine et al. 1993)

Ø It had been concluded that, in an animal homozygous for the Gh2 allele, only the GH2-N gene was expressed in the sheep pituitary. (Gootwine et al. 1996)

Ø Sequences of each of the GH1, GH2-N and GH2-Z copies of the ovine GH genes were worked out of Awassi (a fat tailed breed) and Romney breeds of sheep and compared them with DNA sequences of pituitary and placental ovine GH expressed. (Gene Bank Accession numbers AF002110-AF2129) The results showed that the structure of the single GH gene copy of the ovine GH1 allele was highly conserved and the structure of the GH2-N copy of the GH2 allele was slightly divergent from the GH1 copy and was polymorphic, and that the GH2-Z gene copies were also polymorphic and accumulated more substitutions both in the coding and the non-coding regions then the GH2-N gene copy. (Ofir at al. 1997)

Ø Growth hormones expressed in ovine placenta were studied and two GH proteins were found 22- and 28-kD. Sequences for three GH-related cDNAs derived from sheep placenta were also worked out. One of these, coding for a protein identical to pituitary preGH, corresponds to the product of the GH1 or GH2-N gene. The other two were very similar and code for a protein differing from pituitary preGH at four amino acid residues, which appeared to correspond to the product of the GH2-Z gene. (Lacroix et al. 1996)

Ø Two clones that contain goat growth hormone (gGH) genes were isolated from goat genomic library using goat growth hormone cDNA as a probe. One clone CgGH contained gGH1 gene, and another clone, EgGH, contained gGH2 and gGH3 genes in tandem. DNA fragments containing gGH1 gene, and that containing gGH2 and gGH3 genes, were estimated to be allelic on the goat chromosome. (Yamano et al. 1991)

3.2 GH gene duplication in animals other than caprine ruminants

Ø The nucleotide sequence of three non-allelic genomic DNA fragments which each contain one member of growth hormone gene had been done. These genes code for the known polypeptide hormones, growth hormone (hGH), chorionic somatomammotropin (hCS), and a yet unknown protein, which differs from hGH in 13 positions. Each gene is structured into five exons, the four introns occurring at identical positions, reflecting recent gene divergence. (Seeburg PH. 1982)

Ø The structure of the human growth hormone gene cluster had been determined over a 78-kilo base region of DNA by the study of two overlapping cosmids. There are two growth hormone genes interspersed with three chorionic somatomammotropin genes, all in the same transcriptional orientation. Analysis of the sequences of the genes and identification of at least three different classes of duplication units interspersed throughout the five gene cluster suggests that the cluster evolved quite recently and that the mechanism of gene duplication involved homologous but unequal exchange between middle repetitive elements of the Alu family. (Barsh GS et al. 1983)

Ø Six RFLPs were detected in human growth hormone / human somatomammotropin gene cluster, patterns of polymorphism and linkage disequilibrium suggested independent origins of the human growth hormone gene cluster. (Chakravarti A. et al. 1984)

Ø Genomic clones containing the closely related genes for human growth hormone (hGH) and chorionic somatomammotropin (hCS) were obtained from genomic bacteriophage lambda and cosmid libraries. The hGH/hCS locus contains two GH genes and three CS genes spanning 48 kb of DNA. Data obtained from that experiment combined with the nucleotide sequences of all five GH and CS genes, indicated that the hGH/hCS gene locus has evolved by duplication mechanism. (Hirt H. et al. 1987)

Ø The human chromosomal growth hormone locus contained on cloned DNA and spanning approximately 66,500 bp was sequenced, and it was found that this locus evolved by a series of duplications and was found to contain five genes, which displayed 95% sequence identity. The expression of each gene was examined by screening pituitary and placental cDNA libraries by using gene-specific oligonucleotides. According to this analysis, the hGH-N gene was transcribed exclusively in the pituitary, whereas the other four genes (hCS-L, hCS-A, hGH-V, hCS-B) were expressed only in placental tissue. (Chen at al. 1989)

Ø Several isoforms of growth hormone (GH) have been identified in humans. There are many reasons for this heterogeneity. At the genetic level, two genes encode GH: GH-N, expressed in the pituitary, and GH-V, expressed in the placenta. At the mRNA level, GH-N undergoes alternative splicing into 20K and 22K isoforms. Post-translationally, 22K GH undergoes modifications, such as acetylation at its amino terminus, deamidation, and oligomerization. The picture is complicated further in the circulation, where GH binds to two GH-binding proteins, each with different affinities for the GH isoforms. (Baumann G. 1999)

Ø Rhesus monkey pituitary and placental cDNA libraries were screened for hCS-hybridizing clones and found that there are at least five genes that code for GH-like proteins, one expressed in the pituitary and four in the placenta, although these may not be exactly equivalent to the human GH-like genes (Golos et al.1993)

Ø The sequence of two growth hormone encoding genes from tilapia fish (Tilapia nilotica) was reported sequencing data indicated that it was a consequence of a relatively recent duplication event. The two genes were highly homologous, having a similar intron (five)/exon (six) arrangement, and both encode an identical polypeptide. (Ber at al. 1993)

3.3 Sequence studies of growth hormone

Ø The ovine growth hormone gene has been isolated and sequenced, together with about 1 kbp of DNA flanking each end of the gene. The structure of the gene was similar to that found for other growth hormone genes, particularly the bovine gene, and has a primary transcript of 1792 bp, with five exons, and with intron sizes of 264 bp, 231 bp, 227 bp and 273 bp (Byrne CR, at al.1987)

Ø Sequence for the sheep GH gene was worked out and the coding sequence predicted from that corresponded to the cDNA sequence for pituitary GH, and this gene presumably codes for the hormone expressed in the pituitary. (Orian et al. 1988)

Ø cDNA was prepared from the mRNA isolated from sheep anterior pituitary glands. On cloning cDNA in E. coli, a clone coding full sequence of sheep pre-growth hormone was determined. The sequence for the sheep growth hormone (GH) is in agreement with the amino acid sequence of the protein determined previously except positions 99 and 146 (Guron C, at al. 1992)

Ø The cDNA that encodes goat growth hormone (gGH) was isolated from a goat pituitary cDNA library. The cDNA was about 880 base pairs long and could code for a polypeptide of 217 amino acids, The amino acid sequence homology between gGH and the sequences of bovine GH, rat GH and human GH was 99, 83 and 66%, respectively (Yamono Y, at al. 1988)

Ø Cloning and nucleotide sequencing of the bovine growth hormone gene has been done. The sequence was about 1800 bp with four introns. The comparison of untanslated regions of the bovine, human and rat growth hormone genes reveled many areas of highly conserved sequence showing divergent evolution. (RP Woychik, et al.1982)

Ø Cloning and characterization of the rabbit growth hormone-encoding gene had been done. The amino acid sequence of rabbit GH is similar to that of pig GH and other conserved mammalian GH, but differs markedly from the available sequences of ruminant and primate GH. No evidence of cluster of GH like genes were found as found in primates. This provides further support for the idea that, in mammals, GH shows a slow underlying rate of evolution. (Wallis OC & Wallis M. et al. 1995)

3.4 Physiological effects of GH copy number.

Ø Growth hormone (GH) gene expression was investigated in pituitaries of lambs from flocks selected for high (fat) or low (lean) back fat depth, which were also homozygous for a single GH gene allele, heterozygous or homozygous for duplication in the GH gene. It was conclude that the pituitary glands of lean sheep are bigger and have an increased GH content, but appear to contain similar concentrations of GH mRNA and immunoreactive GH as the pituitaries of fat sheep. The presence of the GH gene duplication in sheep has little measurable effect on the expression and storage of GH in the pituitary. (Fleming at al.1997)

Ø The effect of growth hormone gene copy number (two three or four for different genotypes of GH gene) on growth parameters, plasma GH profile and response to GHRH were studied. No significant effect of GH genotype on any growth parameter or body composition was found. But Significant differences were found between GH genotype and response to GHRH, this suggested that polymorphism in GH gene copy number may have its physiological implications for the functioning of the GH axis. (Gootwine at al. 1997)

3.5 Structural functional aspects of duplicate GH gene

Ø The sequence of the second GH expressed in the sheep placenta (probably the product of the GH2-Z gene) was worked out and it was found that it differed from pituitary GH at three sites, one (Pro®Leu at position7) in the signal peptide, and two (Gly®Arg at position 9 and Gly®Ser at position 63). The numbering is based on N-terminal Ala as residue 1. The second placental GH-like protein that they observed was substantially larger than normal pituitary GH. (Lacroix et al. 1996)

Ø The substitution at residue 9 is in the second receptor-binding site on GH. For human GH. Conversion of this residue Arg to Ala led to some lowering of receptor binding (Cunningham et al. 1991

Ø The Gly®Arg conversion at position 9, in the sheep placental GH was found to lead to enhanced affinity for the receptor at the second site. This was supported by modeling studies on ovine GH. Introduction of Arg at position 9 would allow formation of a salt bridge and hydrogen bond to Glu 122 and Ser 120 respectively on the receptor, with enhanced binding. (A Lioupis, unpublished observations)

Ø Residue 63 (Gly® Ser substitution in the placental GH) is located in binding site 1 in human GH. The corresponding residue in human GH is Asn, and conversion of this to Ala led to some decrease in receptor binding. (Cunningham & Wells 1989)

3.6 Evolutionary significance of GH gene duplication.

Ø Pituitary growth hormone (GH) and prolactin have been shown previously to display a pattern of evolution in which episodes of rapid change are imposed on a low underlying basal rate (near-stasis). Seven protein hormones were examined. Six of these (GH, prolactin, insulin, parathyroid hormone, glycoprotein hormone alpha-subunit, and luteinizing hormone beta-subunit) showed markedly variable evolutionary rates. Two protein hormones (follicle-stimulating hormone beta-subunit and thyroid-stimulating hormone beta-subunit) showed no significant rate variation. Where gene duplication is associated with a period of accelerated evolution this often occurs at the end rather than the beginning of the episode. (Wallis M 2001)

Ø Cloning and characterization of the gene encoding red-deer (Cervus elaphus) growth hormone showed the molecular evolution of growth hormone in artiodactyls. In mammals the structure of pituitary GH is generally strongly conserved, indicating a slow basal rate of molecular evolution. However, on two occasions, during the evolution of primates and of artiodactyls, the rate of evolution has increased dramatically (25--50-fold). Differences between the signal peptide sequences of red deer and bovid GHs probably explain why N-terminal heterogeneity is seen in bovine, ovine and caprine GHs but not GH from red deer, pig or most other mammals. (Lioupis A, et al. 1997)

Ø The growth hormone-prolactin gene family in mammals is an interesting example of evolution by gene duplication. Divergence among duplicated genes is characterized by the relatively high rate of non-synonymous substitutions. So pattern of nucleotide substitution in growth hormone-prolactin gene family provided a paradigm for evolution by gene duplication. (Ohta T. et al. 1993)

4. Justification and likely benefits

4.1 Specific benefits

1) Work has not been done on Pakistani breeds of sheep, so by the completion of this project we can know that is there any duplication of GH gene is present in Pakistani breed of sheep or not, this would be more useful when the complete physiological role of GH gene duplication would be known.

2) The success full completion of this project can lay a foundation for recombinant DNA work on GH of Pakistani fat tailed sheep, as gene mapping could be done so this information would be a starting point for any work on GH gene of sheep.

3) The project would be useful to sort out the evolutionary position of sheep.

4) It would confirm the findings of other people about GH gene duplication in sheep.

4.2 General benefits.

1) It may help to understand molecular evolution and animal production.

2) It may develop a PCR based method in institute of biochemistry and biotechnology.

5. Objectives

For PCR based method, objectives are

1) Probe designing.

2) Amplification of growth hormone genes.

3) RFLP and restriction mapping of amplified genes.

For southern blotting and GH mRNA probe hybridization-based method, objectives are.

1) Formation of growth hormone mRNA non-radiolabled probe.

2) Southern blotting of genomic DNA restriction digest, with mRNA probe.

6. Plan of work and methodologies

To study growth hormone gene duplication in sheep several methods can be used, these are (1) PCR amplification of GH genes (2) screening of sheep genomic DNA library or cDNA libraries of sheep placenta and pituitary, using growth hormone mRNA as probe (3) RFLP based studies, using probe for GH gene. In this synopsis only two techniques PCR and RFLP based are given as the construction of genomic or cDNA libraries is a very time consuming and difficult task further more the genotypic differences in GH gene copy number further complicates the conditions. The first option would be PCR based study as it gives reliable results; it is faster and fits the time given for a Msc research. A pale alternative is a RFLP based method in which genomic DNA would be digested and southern blotting would be performed using GH mRNA as probe. The problem with this technique is that it doesn’t give reliable results; some people observed bovine GH gene duplication but such results are not reproducible with the use of other techniques.

6.1 PCR based method

Polymerase chain reaction can be used to study GH gene duplication and use of this technique would be the first option. The technique has been used successfully to study GH gene duplication in Awassi and Romany breeds of sheep (Offir and Gootwine, 1997).

6.1.1 Primer designing

PCR primers are short oligonucleotides that are designed to compliment the end sequences of the target sequence to be amplified. To amplify GH genes a primer pair would be designed complimentary to the conserved sequence of growth hormone gene. As conserved sequences exist in coding regions and sequence alignments has been done for about 15 different mammals (http://www.biols.susx.ac.uk/Home/Mike_Wallis/GHAlign) using these sequences and appropriate primer design software primer can be designed. Some points that would be considered in primer design are (1)- Primer length is usually 15 to 25 nucleotides. (2)- G + C content should be ~50% (3)- The primer pair should not be complimentary (especially in3¢ ends) to each other to form duplex or to form hairpin loops within themselves. (4)- The 3¢ end of primer should match the target.

6.1.2 Melting and annealing temperature prediction.

Tm is a temperature at which half of DNA molecules are single stranded, it depends on a number of factors like G + C content of duplex, salt concentration of buffer and is also different for both primers so a any appropriate software would be used to calculate Tm. Ta is the annealing temperature of target DNA and primer and is also calculated using software it is usually 5° below Tm. Even when all parameters are calculated amplification is not always up to the mark so several possibilities need to be tried before getting desired results.

6.1.3 Collection of spleen samples

Spleen samples would be collected for genomic DNA isolation, as DNA isolated from spleen is comparatively clean. Spleen samples of fat tailed sheep would be collected from local slaughterhouses early in morning so that samples can be preserved fresh. As growth hormone gene duplication is an allelic phenomenon and different animals have different GH copy number i.e. two three or four so all the samples would be collected and further studied separately. The sample size would be sufficiently large so that we can come across at least on such animal in which GH duplication is present. Gloves would be worn in order to prevent DNAase activity from skin. Also new pair of gloves would be worn for each animal, so to prevent the mixing of any DNA. The samples would be frozen to prevent spoilage.

6.1.4 Isolation of genomic DNA

Genomic DNA would be isolated from spleens of different animals separately; as DNA would be isolated for PCR so high purity is not required further more any chemical which can interfere with PCR would not be used in isolation procedure. The most commonly used method of isolating genomic DNA is Blin and Stafford (1976) method; it yields high molecular weight DNA suitable for many purposes including PCR. Alternatively a non-enzymatic method by Lahiri and Nurnberger (1991) can be used, which yields 50 kbp DNA fragments suitable for PCR. Outline of only Stafford method is given hare.

First of all the sample would be homogenized, in cold conditions using a blender then extraction buffer of EDTA, RNase and SDS would be added. The SDS lyses cells and assist the removal of proteins from DNA and inactivates other enzymes like DNases. Proteinase K would be then added to deprotenises the solution. Residual proteins and lipids could be removed by extraction with phenol and chloroform. Finally the DNA can be precipitated and spooled using ethanol. Absorbance of DNA solution would be measured to calculate its purity and A260/A280 ratio should be >1.8.

6.1.5 Amplification

For PCR amplification a programmable thermal cycler would be used and it would be programmed for melting temperature Tm, annealing temperature Ta, time durations and number of cycles. The reagents for PCR include MgCl₂ (the concentration of Mg ions is very important as Mg ions are cofactor of DNA polymerase), the four dNTPs (consisting of dATP, dGTP, dCTP, dTTP) and the two primers. The polymerizing enzyme would be Taq polymerase. In addition of these basic reagents some other reagents may be used for PCR like for “hot start” (in this method the amplification starts at a higher temperature so to prevent misalignment of primer) PCR a wax of appropriate make could be used. Similarly to prevent amplification of contaminating DNA of previous PCR dUTP could be used instead of dTTP (pre treatment of PCR reaction with uracil N-glycoslase would destroy any PCR product from previous reaction). The PCR is usually not always successful, so may be for the first time a smear would appear (excessive amplification), or on other hand no amplification so in either case the reaction conditions would be changed to get the desired amplification. Strict precautions would be observed during PCR so to prevent DNA contaminations.

6.1.6 Electrophoresis of amplified DNA

The PCR products would be analyzed using Agarose gel electrophoresis. For this proposes Agarose gel of appropriate concentration, would be prepared. Ethidium bromide would be added in gel to see the DNA bands. The PCR reaction sample would be taken and mixed with gel loading buffer and would be loaded in wells formed on gel along with size markers. The size marker would be formed by digesting any well-known plasmid with appropriate restriction enzyme; as it is a cheap alternative of commercially available size markers. Running the gel on appropriate voltage would result in several bands like amplified fragment bands, primer dimmer and bands of genomic DNA used. By comparing the sizes of bands the amplified GH gene fragments would be identified and further studied. As the duplicate GH genes differ from pituitary expressed GH gene so there is a possibility that there would be two amplified band, if a single amplified band would be appear then we would go for RFLP studied of amplified product.

6.1.7 RFLP of amplified product

As we know that there is a PVUII polymorphism in the second intron of GH gene (Gootwine at al, 1993) it means that some GH genes contain a PVUII restriction site and other would not have this. So when we would cut PCR product with PVUII, and run the restriction digest on gel, if the previously studied amplified band would be missing and two smaller bands appeared instead, it means that GH gene duplication is not found in that particular animal and if the original larger amplified band would be also present along with two smaller bands it shows duplication. Similarly all the animal samples would be studied and in some animals hopefully GH gene duplication would be encountered.

6.1.8 Restriction mapping and gene alignments

As we know the sequences of GH from about 50 species (available at www.ncbi.nlm.nih.gov/Entrez) 15 of which are mammals. So we could design a restriction-mapping project of our amplified GH genes. We can prove that the conserved sequences present in GH of other mammals are also present in Pakistani fat tailed sheep. During this analysis we may encounter some unique sequence present only on Pakistani breed of fat tailed sheep. For this purpose several available restriction enzymes and their combinations would be used to cut the DNA. The restriction sites so elucidated can be aligned with the known sequences of GH gene.

6.2 Southern blotting and hybridization based method.

The main steps in this technique would be as follow

6.2.1 Isolation of total genomic DNA from Spleen

The isolation of high quality cellular DNA is often a starting point for a variety of molecular-biology techniques. These include Southern-blot analysis, PCR amplification, RFLP and genomic-library construction. Generally, quality is indicated by the absence of contaminating RNA, proteins, lipids, and other cellular constituents that may interfere with restriction enzymes, ligases, and thermostable DNA polymerases. More importantly, the preparation should be free of contaminating DNA nucleases, which can nick and degrade high molecular weight DNA. There are many methods to isolate total cellular DNA. Commonly used procedures employ a buffer containing one or several detergents; for example, SDS, NP-40, or Triton X-100. These detergents lyse cells and assist in the removal of proteins from the DNA. More thorough deproteinization is achieved by the use of proteinase K in the lysis buffer. Hence, the combination of proteinase K, the buffer components, and the elevated temperature are important for the inactivation of endogenous nucleases, in the disruption of cellular integrity and in the release of DNA from the nuclear compartment. DNases are dependent on Mg²⁺and Ca²⁺ for activity. EDTA (at least 2 mM) included in the DNA buffer chelates these cations and thereby prevents the degradation and random nicking of high molecular weight DNA by DNases. To isolate total cellular DNA, Blin and Stafford method would be used.

6.2.2 Collection of Pituitary samples

Pituitary is a small ovoid gland, about the pea size located at the base of the brain, occupies a depression of the sella turcica in the floor of the cranium. Pituitaries of local strains of fat tailed sheep would be collected from slaughterhouse in Lahore. First the skull will be cut open with an axe. Then the brain of the animal will be removed by cutting the connective tissue between the brain and pituitary from the head of the animal. Then after cutting the bony layer, which covers the pituitary with a sharp razor blade, the pituitary will be taken out with the help of a spatula. The samples will be stored at –20^oC in order to prevent spoilage of samples.

6.2.3 Extraction of total RNA from pituitary

There are many methods to isolate total RNA from the tissue. Generally, the rationale for any isolation procedure is to solubilize cellular components and simultaneously inactivate intracellular RNases while maintaining biologically active RNA. Most isolation procedures combine the use of one or more agents, such as organic solvents (i.e., phenol, chloroform), detergents (i.e., SDS, NP-40, sodium deoxycholate), or chaotropic salts, such as guanidium isothyocyanate (GITC), or urea. Often beta-mercaptoethanol is added to further assist in protein denaturation. Phase-Extraction method would be used which capitalizes on the ability of GITC to denature proteins and inactivate intracellular RNases. In this method, an acidic phenol extraction (at pH<5.0) selectively retains cellular DNA in the organic phase. The addition of chloroform further removes lipids and establishes two distinct phases: an organic phase containing the DNA, proteins, and lipids, and an aqueous phase containing the total RNA.

6.2.4 Isolation of mRNA from the total RNA by Affinity Chromatography

Messenger RNA (mRNA) comprises approximately 1-5% of total cellular RNA. The presence of a terminal stretch of approximately 200 adenosine residues (the polyA tail) on most eukaryotic mRNA and its ribosomal and transfer RNAs has important practical consequences, as it allows polyadenylated species (mRNA) to be separated from their nonpolyadenylated counterparts (rRNA & tRNA). The basic principle of this technique, involves the affinity selection of polyadenylated mRNA oligodeoxythymidylate (oligo (dT)).

6.2.5 Isolation of GH mRNA from total mRNA

In the pituitary, various genes are expressed at a time. So it is necessary to isolate our desired mRNA i.e. GH mRNA from the rest of mRNAs. For this purpose, total mRNA obtained from above technique will be electrophoresed, using Agarose-gel electrophoresis. Total mRNA will be fractionated according to its size, and would be compare with different size markers and mRNA of GH would be isolated from the gel.

6.2.6 Non-Radioactive labeling of GH mRNA

mRNA of growth hormone (GH) would be labeled non-radioactively in this step. For this purpose biotinylation of GH mRNA will be done. Biotinylation kits are available in the market. After biotinylation, GH mRNA will be used as a probe.

6.2.7 Summary of procedure

First of all, I shall cut genomic DNA of fat tailed sheep with the help of restriction enzymes, and then the agarose-gel electrophoresis of that DNA will be done. Different bands of different length will come. Then I shall do southern blotting by this DNA of the gel will be transferred on nitrocellulose sheet. Then GH mRNA probe will be used to detect the growth hormone gene/genes having a particular sequence that will bind to GH mRNA. If duplication will present, two hybridized bands will come and if duplication would not present, only a single hybridized band will come.

7. Place of work and facilities available.

All the research work would be completed in the labs of institute of biochemistry and biotechnology, university of the Punjab. The facilities required are PCR machine, gel documentation system, electrophoretic and southern blotting apparatus etc which are available in IBB. The primer for PCR would be synthesized from appropriate supplier.

8. References

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