Growth Hormone

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 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 processing. 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 growth promoting functions of GH are also mediated by somatomedins, the insulin like growth factors. The amino acid sequence homology between goat growth hormone and the sequences of bovine GH, rat GH and human GH was 99, 83 and 66%, respectively (Yamano Y, et al. 1988).

Growth Hormone Gene

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. The structure of ovine growth hormone gene is 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. The gene is flanked by artiodactyl-specific middle-repetitive DNA, consisting mainly of elements belonging to the 'C-A3' family of repeated DNA. (Byrne et al. 1987). The bovine growth hormone gene contains approximately 1793 nucleotides. Comparison of the bovine growth hormone gene to the known sequence of the rat and human genes reveals that the coding regions of the three genes are highly conserved. (Gordon et al. 1983)

The sequence of cDNA corresponding to sheep pituitary GH was reported by Warwick & Wallis (1984) and subsequently confirmed (Warwick et al.1989, Lacroix et al. 1996). The protein sequence predicted from this agrees with the known sequence for sheep GH. Orian et al. (1988) presented a sequence for the sheep GH gene; the coding sequence predicted from this corresponds to the cDNA sequence for pituitary GH, and this gene presumably codes for the hormone expressed in the pituitary. This was confirmed by Ofir & Gootwine (1997) who showed that the sequence of the single GH1 gene (Gh1 allele) is identical to the sequence described by Orian et al. (1988) and that the (partial) sequence of the GH2-N gene is also very similar to this, although there is some variation between strains of sheep. Lacroix et al. (1996) described sequences for three GH-related cDNAs derived from sheep placenta. 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 are very similar and code for a protein diverging from pituitary preGH at three amino acid residues, which appears to correspond to the product of the GH2-Z gene. Byrne et al. (1987) provided a rather divergent sequence for a GH-like gene in sheep, which differs at about 44 nucleotides from the sequence of Orian et al. (1988); in some respects this resembles the GH2-Z gene, but the match is far from complete. This may reflect variation between sheep strains of the kind noted by Ofir & Gootwine (1997). Neither Orian et al. (1988) nor Byrne et al. (1987) reported the presence of two GH genes in the gene libraries that they screened. However, it is notable that Southern blotting of DNA from several individual animals led Byrne et al. (1987) to detect a GH-linked RFLP very similar to that which led Valinsky et al. (1990) to conclude that duplicated GH genes are present in some sheep. The situation in goat is less clear. The sequence of goat pituitary GH cDNA was reported by Yamano et al. (1988) and Yato et al. (1988) and the GH gene sequence was described by Kioka et al. (1989). The coding sequence predicted from the gene sequence corresponds to the goat GH cDNA sequence and this gene was subsequently shown to be the gGH1 gene. Sequence information for the gGH2 and gGH3 genes (i.e. the duplicate genes in the second allele) is not yet available.

Growth Hormone 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 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 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. In some instances gene duplication can increase the transcription of any particular type of proteins, as multiple temples are available for transcription, but GH gene duplication has no such effect on growth parameters. Gene duplication should not be confused with gene amplification, as duplication is a process in which any gene forms many copies permanently. 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.

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 somatomammatropin one gene hGH-V for GH variant and one hCS like gene (Chen et al. 1989). In rhesus monkey five genes codes for GH like proteins, one expressed in pituitary and four in placenta (Golos et al.1993). 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.

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.

Significance of GH Gene Duplication

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 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. 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.

Sheep (Ovis aries) Breeds in Pakistan

Selection for wool type, flocking instinct and other economically important traits over the centuries has resulted in more than 200 distinct breeds of sheep occurring worldwide. Modern breeding schemes have also resulted in an increasing number of composite or synthetic breeds that are the result of a crossing of two or more established breeds. In Pakistan about 52 breeds of sheep are known some economically important breeds are Lohi, Lati, Thalli, Kajli, Baluchi, Balkhi, Bibrik, Cholistani, Damani, Waziri, Harnai, Hasht Nagri, Kachhi and Kooka.

Polymerase Chain Reaction

Polymerase chain reaction was first proposed in the early 1970's by H.Ghobind Khorana and his colleagues as a strategy to lessen the labor involved in Chemical synthesis of genes (Kleppe et al 1971) this idea was not practicable at that time as genes had not yet been sequence, thermostable DNA polymerases had not been described and synthesis of primers was more of an art than science.

The technique was independently conceived 15 years later by Kary Mullis and co-workers, who described in vitro amplification of single copy mammalian gene using the Klenow fragment of E coli DNA polymerase I (Saiki et al. 1988, Mullis et al. 1986, Mullis and Faloona 1987). PCR was a labourous technique before the discovery of thermostable DNA polymerases (Chien et al. 1976, Kaledine et al. 1980). The use of thermostable polymerase from Thermus aquaticus (Saiki et al. 1988) greatly increased the efficiency of PCR and opened the door to automation of the method.

Thermostable DNA Polymerase

The essential components of polymerase chain reaction is a thermostable DNA polymerase, to catalyze template dependent synthesis of DNA. For this purpose a wide choice of enzymes is now available that very in their fidelity, efficiency and ability to synthesize large DNA products. For routine PCR Taq polymerase is used, Taq polymerase is isolated from an organism Thermus aquaticus of thermophilic Archaea family. It was the first isolated and best understood of thermostable DNA polymerases, but unfortunately preparations of Taq polymerase sold by different manufacturers are not identical and it is therefore important to optimize PCRs every time for each new batch of Taq. 0.5-2.5 units of Taq polymerase are required for a standard 25-50 µl reaction. Other polymerases are used when greater Fidelity is required, when the length of the target amplicon exceeds a few thousand bases or when cloning mRNA by reverse transcriptase PCR (RT-PCR), other thermostable enzymes may have significant advantages.

Primers

A pair of synthetic oligonucleotides is also required to prime DNA synthesis. Of the many factors that affect PCR none is more crucial than the design of primers. Careful design of primers is required to obtain the desired products in high yield. Oligonucleotide primers synthesized on an automated DNA synthesizer can generally be used in standard PCR without further purification however amplification of single copy sequences from mammalian genomic template is often more efficient if the nucleotide primers are purified by chromatography on commercially available resins.

dNTPs

Standard PCR contains equimolar amounts of dATP, dTTP, dCTP and dGTP. Concentration of 200 to 400 µM of each dNTP is recommended for Taq polymerase in reactions containing 1.5 mM MgCl₂. dNTP stocks solution should be stored at -20º C and aliquots should be discarded after a few freeze thaw cycles.

Divalent Cations

All thermostable DNA polymerases required free Divalent cations usually Mg²⁺ for their activity. As dNTPs and oligonucleotides bind Mg²⁺, the molar concentration of the cations must exceed the molar concentration of phosphate groups contribute by dNTPs and primers. It is therefore impossible to recommend a concentration of Mg²⁺ that is optimal in all circumstances. At a final dNTP concentration of 0.4mM, MgCl₂ concentration ranges of 2.3 ± 0.25 mM in traditional PCR buffer and of 3.0 ± 0.5 mM in PCR buffer with (NH₄)₂S0₄ are suitable in most cases. Increasing the concentration of Mg²⁺ to 5mM or 6mM has been reported to decrease nonspecific priming in some cases and to increase in others (Harris and Jones 1997).

pH of Reaction Mixture

To maintain the pH of PCR reaction mixture tris-Cl adjusted to a pH between 8.3 and 8.8 at room temperature is included in standard PCR at a Concentration of 10mM. When incubated at 72ºC the pH drops to 7.2, suitable for Taq polymerase.

Template DNA

Template DNA containing target sequence is another important component of PCR reaction mixture. To amplify a single copy DNA segment from a mammalian genomic DNA 1µg DNA is required for a 50µl reaction. Nearly all-routine methods are suitable for template DNA purification although even trace amounts of agents used in DNA purification procedures (phenol, EDTA, Proteinase K etc.) strongly inhibit Taq polymerase. The purity of DNA is not critical but for a protein containing DNA spermidine use is sometime a miracle cure. Although the size of template DNA is not critical, amplification of sequences imbedded in high molecular weight DNA (>10 kb) can be improved by digesting the template with a restrictions enzyme that does not cleave within the target sequence. On the other hand to amplify a larger DNA segment highly fragmented template is not recommended.

Temperature Cycling

PCR is an interactive process, consisting of three elements: denaturation of the template by heat, annealing of the primers to the single stranded target sequence, and extensions of the annealed primers by a thermostable DNA polymerase.

The first step in temperature cycling is the initial denaturation of the DNA template, incomplete denaturation of template DNA results in the insufficient utilization of template in the first amplification cycle and in a poor yield of PCR product. The initial denaturation should be performed over an interval of 1-3 min at 95ºC if the GC content is 50% or less. This interval should be extended up to 10 min for higher GC content templates. Some authors claim that initial denaturation step is unnecessary for linear DNA molecule and may sometimes be deleterious (Gustafson et al. 1993). Denaturation temperature is determined in part by the GC content of double stranded DNA the high the proportion of GC the higher the temperature required to separate the strands. The longer the DNA molecules, the greater the time required at the chosen denaturation temperature.

Denaturation step; usually 0.5-2 min denaturation at 94–95ºC is sufficient, since the PCR product synthesized in the first amplification cycle is significantly shorter than the template DNA and is completely denatured under these conditions. If the amplified DNA has a very highly GC content the time may be increased up to3-4 min or additives may be added to facilitate DNA denaturation.

Primer annealing step; usually the optimal annealing temperature is 5ºC lower than the melting temperature of primer template DNA duplex. Incubation of 0.5-2 min is sufficient. Annealing temperature is critical if it is too high the primers anneal poor if at all, to the template and the yield of amplified DNA is very low. If the annealing temperature is too low, nonspecific annealing of the primers may occur, resulting in mispriming.

Extension step; usually the extension step is performed at 70 to 75ºC. The rate of DNA synthesis by Taq is highest at this temperature (2-4 kb/min), and a one min extending time is sufficient for this synthesis of PCR fragments up to 2kb.

The number of PCR cycles depends on the amount of template DNA in the reaction mixture. If template quantity is high 25 - 35 cycles are sufficient. Final extending step; after the last cycle the samples are usually incubated at 72ºC for 5-15 min to fill in the protruding ends of newly synthesized PCR products.

Restriction of PCR Product

Restriction enzymes were discovered about 40 years ago during investigations into the phenomenon of host-specific restriction and modification of bacterial viruses. (Arber, W. and Dussoix, D. 1962). In 1968, restriction modification enzymes EcoB and EcoK were isolated and classified as type I enzymes. (Linn S. and Arber S. 1968, Meselson M. and Yuan R. 1968). Two years later Smith and Wilcox (1970) isolated and characterized the first type II restriction endonuclease, HindII that cleaved DNA in well defined fragments. This discovery revolutionized research into gene structure and gene expression. More than 3000 type II restriction endonucleases, exhibiting 233 different specificities, have been isolated so far. Type II R-M system enzymes recognize nucleotide palindromes 4-8 bp in length, interrupted palindromes with some unspecific nucleotides between flanking nucleotides or partially palindromic sequences with ambiguous nucleotides at certain positions. For most nucleotide sequences, more than one enzyme is available that recognizes that sequence. The restriction enzymes require only Mg²⁺ for activity and cleave DNA within the recognition sites, leaving 5’-P and 3’-OH termini.

Restriction enzymes are traditionally classified into three types on the basis of subunit composition, cleavage position, sequence-specificity and cofactor-requirements. Type I enzymes are complex, multisubunit and cut DNA at random far from their recognition sequences. Type II enzymes cut DNA at defined positions close to or within their recognition sequences. These are the enzymes used in DNA restriction studies and principle ones available commercially. Type III enzymes are also large combination restriction-and-modification enzymes. They cleave outside of their recognition sequences and require two such sequences in opposite orientations within the same DNA molecule to accomplish cleavage.

In experiments with amplified DNA, restriction endonuclease (RE) digestion is usually performed directly in the PCR mixture, without time consuming and expensive purification steps. The majority of restriction enzymes show sufficient activity (>20%) in PCR buffers (traditional PCR buffer and PCR buffer with (NH₄)₂SO₄); some require the addition of their optimal buffer to obtain adequate activity. The one unit of enzyme activity is defined as the amount needed to digest 1µg of a specific DNA in a 50µl digest at the appropriate temperature (usually 37ºC) in one hour, but many factors like DNA purity effects digestion and hence over digestion is required (Increased enzyme and or increased incubation time). It has been shown that three sequential NH₄Ac:EtOH precipitations reduced the amount of enzyme needed 10 fold over no precipitation.

None of the PCR mixture components, including primers, dNTP's, template DNA and Taq DNA Polymerase affect RE activity. In some cases, a RE having 100% activity in the initial PCR mixture is not able to digest the PCR product, even after addition of extra amounts of the RE or its optimal buffer. There have been reports in the literature on the inability of some REs to digest DNA following PCR, however, these data are contradictory (Blanck, A., et al. 1995, Eastlake, P., et al. 1995). Enzymes from different companies differs in their ability to digest the PCR product, depending on the nature of template DNA and primers used. In these cases, thorough purification of the PCR product may help. In some cases a large excess of enzyme or prolonged incubation time is necessary.

Another factor to be considered is that some restriction enzymes cleave DNA poorly when their recognition sites are close to the end of the DNA fragment. This problem is very common when working with PCR product. The cleavage ability at the termini differs for different enzymes but usually 100% activity is obtained if recognition sequence is 3-4 bases away from the termini of PCR product.

Restriction Mapping of Genes

The treatment of a DNA molecule with a restriction enzyme produces a series of precisely defined fragments that can be separated according to size by gel electrophoresis. Using this technique relative positions of cleavage sites of different restriction enzymes on a DNA fragment can be found, a diagram showing the position of restriction sites of different enzymes on a DNA molecule is called a restriction map.

The most straightforward method for restriction mapping is to digest DNA of interest with a set of individual enzymes, and with pairs of those enzymes. Alternatively if a fragment of DNA is labeled with a radioisotope on only one end, it can be partially digested with single restriction enzymes to generate labeled fragments that directly reveal where the cleavage sites are located. Kenneth D. Bloch 1999.

Restriction Sites of the Enzymes Used in the Study

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ApaI 5’…..G G G C C C….3’

3’.….C C C G G G….5’

↑

↓

Bsp1431 (MboI) 5’…..G A T C….3’

3’.….C T A G….5’

↑

↓

BsuRI (HaeIII) 5’…..G G C C….3’

3’.….C C G G….5’

↑

↓ _*

MvaI (EcoRII) Star activity 5’…..C C W G G….3’

3’.….G G W C C….5’

↑

↓

PstI 5’…..C T G C A G….3’

3’.….G A C G T C….5’

↑

↓

PvuII (Star activity) 5’…..C A G C T G….3’

3’.….G T C G A C….5’

↑

↓

SmaI (Star activity) 5’…..C C C G G G….3’

3’.….G G G C C C….5’

↑

* W stands for A or T