Genomic Structure and Promoter Analysis of the Bubalus Bubalis Leptin Gene

The buffalo is an animal ever-growing in our hemisphere, one of the major problems for the genetic improvement of this species, concerns two reproductive aspects: the difficult heat detection and the seasonality of the oestrus cycles. The leptin plays a critical role in the regulation of reproductive and immune function in humans, it is at the centre of the complex networks that coordinate changes in nutritional state with many diverse aspects of mammalian biology. We have sequenced the 5’ flanking region and exon 1 of the leptin gene in buffalo. The sequencing is the 'first step' for understanding the role of various parts of the genome and is a springboard from which to decode the genome. The simple sequencing, in fact, does not provide information directly applicable to understand the mechanisms underlying physiological and pathological processes, but represents a necessary step through which you can identify the role of different regions of DNA. However, waiting for the complete genome sequence of the buffalo, the database of the bovine genome offers the opportunity to investigate the buffalo genome in genes which are recognized to influence physiological processes related to reproduction in other species. In this context, our research had decided to investigate the leptin gene and particularly the regulatory area: the promoter.


INTRODUCTION
The buffalo is an animal ever-growing in our hemisphere, both for its greater rusticity, longevity, better adapted to humid environments, and for the highest income generated from the sale / transformation of the milk, that is not subject to quotas in European countries.
However, one of the major problems for the genetic improvement of this species, concerns two reproductive aspects: the first is the difficult heat detection, caused by silent heats, that is an obstacle to the widespread use of instrumental insemination; the second is the seasonality of the oestrus cycles, because buffaloes go into heat between October and December.
The next -generation sequencing technologies have already contributed to the characterisation of farmed animal genomes, and the development of high throughput single nucleotide polymorphisms (SNP) genotyping platforms and the first applications in animal breeding have begun to emerge.Breeding and selection of cattle, pig and sheep have started to take into account genomic information to some extent.In the next few years it is expected that also the buffalo genome will be sequenced, and it will be possible to deliver genomics-driven improvement in buffalo breeding and production.
*Address corresponding to this author at the CRA-PCM, Via Salaria 31, 00015 Monterotondo (Rome), Italy; Tel: +39 06 90090215; Fax: +39 06 9061541; E-mail: francesco.napolitano@entecra.itIn fact, the sequencing is the 'first step' for understanding the role of various parts of the genome and is a springboard from which to decode the genome.The simple sequencing, in fact, does not provide information directly applicable to understand the mechanisms underlying physiological and pathological processes, but represents a necessary step through which you can identify the role of different regions of DNA.
However, before the complete sequence of the buffalo genome is available and considering the high sequence homology between cattle and buffalo (96-97% for the coding and regulatory regions of genes), the database of the bovine genome offers the opportunity to investigate the buffalo genome in genes (candidate genes) which are recognized to influence physiological processes related to reproduction in other species.In this context, our research had decided to investigate the leptin gene and particularly the regulatory area: the promoter.
In genetics, a promoter is a region of DNA that facilitates the transcription of a particular gene.Promoters are located near the genes they regulate, on the same strand and typically upstream of the transcription start site.In order for the transcription to take place, the enzyme that synthesizes RNA, known as RNA-polymerase, must attach to the DNA near a gene.Promoters contain specific DNA sequences and response elements which provide a secure initial binding site for RNA polymerase and for proteins called 'transcription factors' that recruit RNA polymerase.
These transcription factors have specific 'activator' or 'repressor' sequences of corresponding nucleotides that attach to specific promoters and regulate gene expressions.
The leptin gene was identified in 1995 as the product of the obese gene and a hormonal signal that regulates energy balance in mice.In human, Farooqi and O'Rahilly [1] have defined the role of leptinresponsive pathways in the regulation of eating behaviour, intermediary metabolism, and the onset of puberty.They also demonstrated that leptin signaling plays a critical role in the regulation of reproductive and immune function in humans, which places leptin at the centre of the complex networks that coordinate changes in nutritional state with many diverse aspects of mammalian biology.
The leptin gene is highly conserved across species and is located on chromosome 4q32 in the bovine [2].Taniguchi et al. [3] have isolated a bovine genomic clone that contained about 3-kb in 5'-flanking region upstream from the putative transcription start site (Figure 1).

EXPONENTIAL AMPLIFICATION BY PCR (POLYMERASE CHAIN REACTION)
Starting from the above Bos taurus sequence (Figure 1), two amplicons were designed: red amplicon, 1141 bp in size and blue amplicon, 613 bp in size; so to cover the 5' flanking and exon 1 of the leptin gene, as indicated by Taniguchi et al. [4] (GenBank: AB070368).
Polymerase Chain Reaction allows us to synthesize several times (amplification) by an enzyme a specific segment of DNA located between two regions of known nucleotide sequence, producing a large number of copies through a series of reactions: -denaturation (95°C) -annealing of primers (40-68°C) -polymerization of new fragments (amplicons) by Taq-Polymerase (72 °C).
The reaction is carried out in a Thermal Cycler (Figure 2).The amplification products are visualized by agarose gel electrophoresis (Figure 3).DNA fragments migrate through a material selective (e.g.agarose gel) that separates them by size (molecular weight / length); smaller fragments migrate through the meshes of the gel faster than larger ones, which move more slowly.DNA of 41 non related buffaloes was PCR amplified and sequenced.Direct sequencing by capillary electrophoresis was then performed using the Big Dye terminator v3.1 on Applied Biosystems 3500 Genetic Analyzer (Figure 5).

CAPILLARY ELECTROPHORESIS
The principle of electrophoresis is the same: the DNA fragments migrate through a resin selective within a capillary that separates them by size (molecular weight / length), smaller fragments migrate through the meshes of the resin faster, first out from the capillary and are intercepted first by the detection system.The detection system consists of a light beam that strikes the DNA fragments.
If marked with fluorescent molecules, the DNA fragment sends an output signal that is recorded by the system.
The result is an electropherogram (Figure 6): The electropherogram of the same piece for more subjects allows us to obtain the complete sequence of the fragment and detected eventual variations (SNPs ).
SNPs are a class of molecular markers (differences due to mutations of homologous DNA regions in different individuals of the same species or different species) whose main features are:

Figure 3 :
Figure 3: Gel electrophoresis apparatus -An agarose gel is placed in this buffer-filled box and electrical field is applied via the power supply to the rear.The negative terminal is at the far end (black wire), so DNA migrates toward the camera.

Figure 6 :
Figure 6: Electropherogram of multiple samples and SNP detection.