Tyrosinase is a bifunctional enzyme which oxidizes the initial step of melanin biosynthesis, that is, conversion of tyrosine to dopa and subsequently dopa to dopaquinone. It is a glycosylated protein and a major regulator of melanogenesis. To date, many approaches have been tried to regulate tyrosinase activity and melanin content. To that end, we screened small interfering RNA sequences for sequence-inhibited tyrosinase expression in B16 cells and in C57BL/6 mice. We analyzed tyrosinase mRNA levels by quantitative real-time PCR and determined tyrosinase activity and melanin content at 24, 48, and 72 hours after transfection. Results showed that siNM_011661_001 was the most efficient small interfering RNA sequence in suppressing tyrosinase mRNA expression, and cells transfected with this sequence showed lower tyrosinase activity. Moreover, intravitreous injection of siNM_011661_001 in C57BL/6 mice induced an efficient and stable gene-specific inhibition of expression at the posttranscriptional level.

Jia Xiu-Hua

Lin Shao-Chun

Huang Bing

Zhu Xiang

Zhuang Jing

Li Wei-Hua

Liu Qian

Luo Ting

Xu Xiao-Ping

Chen Xi-Gu

Tyrosinase Small Interfering RNA Effectively Suppresses Tyrosinase Gene Expression In Vitro and In Vivo

We quantified chicken <i  >calpain 2</i> (<i  >CAPN2</i>) expression in two Chinese chicken breeds (mountainous black-bone chicken breed [MB] and a commercial meat type chicken breed [S01]) to discern the tissue and ontogenic expression pattern and its effect on muscle metabolism. Real-time quantitative PCR assay was developed for accurate measurement of the <i  >CAPN2</i> mRNA expression in various tissues from chickens of different ages (0, 2, 4, 6, 8, 10, and 12 weeks). Results showed that the breast muscle and leg muscle tissues had the highest expression of <i  >CAPN2</i> compared to the other tissues from the same individual (<svg     style="vertical-align:-0.1638pt;width:50.549999px;" id="M1" height="11.375" version="1.1" viewBox="0 0 50.549999 11.375" width="50.549999"  xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg">
	
		
			<g transform="matrix(.017,-0,0,-.017,.062,11.112)"><path id="x1D443" d="M619 482q0 -69 -40.5 -119t-96 -72.5t-118.5 -26.5h-44l-70 20l-31 -151q-14 -67 0.5 -83t89.5 -22l-5 -28h-287l8 28q65 7 81.5 22t29.5 83l79 398q12 56 0.5 70.5t-78.5 20.5l7 28h255q108 0 164 -43t56 -125zM524 478q0 141 -146 141q-25 0 -47 -8q-16 -6 -20.5 -13.5&#xA;t-10.5 -39.5l-43 -241q37 -13 83 -13q67 0 125.5 45t58.5 129z" /></g><g transform="matrix(.017,-0,0,-.017,15.582,11.112)"><path id="x3C" d="M512 -3l-437 233v51l437 233v-58l-378 -200v-2l378 -199v-58z" /></g><g transform="matrix(.017,-0,0,-.017,30.286,11.112)"><path id="x2E" d="M113 -12q-24 0 -39.5 16t-15.5 42q0 24 16 40.5t40 16.5t40 -16.5t16 -40.5q0 -26 -16 -42t-41 -16z" /></g><g transform="matrix(.017,-0,0,-.017,34.162,11.112)"><path id="x30" d="M241 635q53 0 94 -28.5t63.5 -76t33.5 -102.5t11 -116q0 -58 -11 -112.5t-34 -103.5t-63.5 -78.5t-94.5 -29.5t-95 28t-64.5 75t-34.5 102.5t-11 118.5q0 58 11.5 112.5t34.5 103t64.5 78t95.5 29.5zM238 602q-32 0 -55.5 -25t-35.5 -68t-17.5 -91t-5.5 -105&#xA;q0 -76 10 -138.5t37 -107.5t69 -45q32 0 55.5 25t35.5 68.5t17.5 91.5t5.5 105t-5.5 105.5t-18 92t-36 68t-56.5 24.5z" /></g><g transform="matrix(.017,-0,0,-.017,42.321,11.112)"><path id="x35" d="M153 550l-26 -186q79 31 111 31q90 0 141.5 -51t51.5 -119q0 -93 -89 -166q-85 -69 -173 -71q-32 0 -61.5 11.5t-41.5 23.5q-18 17 -17 34q2 16 22 33q14 9 26 -1q61 -50 124 -50q60 0 93 43.5t33 104.5q0 69 -41.5 110t-121.5 41q-53 0 -102 -20l38 305h286l6 -8&#xA;l-26 -65h-233z" /></g>

		
	
</svg>). Overall, the <i  >CAPN2</i> mRNA level exhibited a &#x201c;rise&#x201d; developmental change in all tissues. The S01 chicken had a higher expression of the <i  >CAPN2</i> mRNA in all tissues than the MB chicken. Our results suggest that chicken <i  >CAPN2</i> expression may be related to chicken breeds and tissues.

Zeng-Rong Zhang

Xiao-Song Jiang

Hua-Rui Du

Xiao-Cheng Li

Qing Zhu

Yi-Ping Liu

Tissue-Specific Expression of the Chicken <i>Calpain2</i> Gene

Molecular defects responsible for <svg style="vertical-align:-2.29482pt;width:8.8500004px;" id="M2" height="13.425" version="1.1" viewBox="0 0 8.8500004 13.425" width="8.8500004"  xmlns="http://www.w3.org/2000/svg">
	<g transform="matrix(1.25,0,0,-1.25,0,13.425)">
		<g transform="translate(72,-61.26)">
			<text transform="matrix(1,0,0,-1,-71.95,63.6)">
				<tspan style="font-size: 12.50px; " x="0" y="0">𝛽</tspan>
			</text>

		</g>
	</g>
</svg>-thalassemias (thal) were investigated among 254 chromosomes from 127 transfusion-dependent unrelated thalassemic patients from two provinces in Northern Iraq. Among fourteen identified mutations, the seven most common found in 88.2&#x25; of the thal chromosomes were: IVS-II-1 (<svg style="vertical-align:-0.17554pt;width:51.0625px;" id="M3" height="10.9125" version="1.1" viewBox="0 0 51.0625 10.9125" width="51.0625"  xmlns="http://www.w3.org/2000/svg">
	<g transform="matrix(1.25,0,0,-1.25,0,10.9125)">
		<g transform="translate(72,-63.27)">
			<text transform="matrix(1,0,0,-1,-71.95,63.5)">
				<tspan style="font-size: 12.50px; " x="0" y="0">G</tspan>
			</text>

			<text transform="matrix(1,0,0,-1,-57.37,63.5)">
				<tspan style="font-size: 12.50px; " x="0" y="0">→</tspan>
			</text>

			<text transform="matrix(1,0,0,-1,-40.23,63.5)">
				<tspan style="font-size: 12.50px; " x="0" y="0">A</tspan>
			</text>

		</g>
	</g>
</svg>), IVS-I-1 (<svg style="vertical-align:-0.17554pt;width:51.0625px;" id="M4" height="10.9125" version="1.1" viewBox="0 0 51.0625 10.9125" width="51.0625"  xmlns="http://www.w3.org/2000/svg">
	<g transform="matrix(1.25,0,0,-1.25,0,10.9125)">
		<g transform="translate(72,-63.27)">
			<text transform="matrix(1,0,0,-1,-71.95,63.5)">
				<tspan style="font-size: 12.50px; " x="0" y="0">G</tspan>
			</text>

			<text transform="matrix(1,0,0,-1,-57.37,63.5)">
				<tspan style="font-size: 12.50px; " x="0" y="0">→</tspan>
			</text>

			<text transform="matrix(1,0,0,-1,-40.23,63.5)">
				<tspan style="font-size: 12.50px; " x="0" y="0">A</tspan>
			</text>

		</g>
	</g>
</svg>), codon 8 (<svg style="vertical-align:-0.0pt;width:33.400002px;" id="M5" height="10.6625" version="1.1" viewBox="0 0 33.400002 10.6625" width="33.400002"  xmlns="http://www.w3.org/2000/svg">
	<g transform="matrix(1.25,0,0,-1.25,0,10.6625)">
		<g transform="translate(72,-63.47)">
			<text transform="matrix(1,0,0,-1,-71.95,63.52)">
				<tspan style="font-size: 12.50px; " x="0" y="0">−</tspan>
			</text>

			<text transform="matrix(1,0,0,-1,-63.39,63.52)">
				<tspan style="font-size: 12.50px; " x="0" y="0">A</tspan>
				<tspan style="font-size: 12.50px; " x="9.0271664" y="0">A</tspan>
			</text>

		</g>
	</g>
</svg>), codon 39 (<svg style="vertical-align:-0.17554pt;width:49.325001px;" id="M6" height="10.9125" version="1.1" viewBox="0 0 49.325001 10.9125" width="49.325001"  xmlns="http://www.w3.org/2000/svg">
	<g transform="matrix(1.25,0,0,-1.25,0,10.9125)">
		<g transform="translate(72,-63.27)">
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				<tspan style="font-size: 12.50px; " x="0" y="0">G</tspan>
			</text>

			<text transform="matrix(1,0,0,-1,-57.37,63.5)">
				<tspan style="font-size: 12.50px; " x="0" y="0">→</tspan>
			</text>

			<text transform="matrix(1,0,0,-1,-40.23,63.5)">
				<tspan style="font-size: 12.50px; " x="0" y="0">T</tspan>
			</text>

		</g>
	</g>
</svg>), codon 8/9 (<svg style="vertical-align:-0.51414pt;width:22.112499px;" id="M7" height="11.325" version="1.1" viewBox="0 0 22.112499 11.325" width="22.112499"  xmlns="http://www.w3.org/2000/svg">
	<g transform="matrix(1.25,0,0,-1.25,0,11.325)">
		<g transform="translate(72,-62.94)">
			<text transform="matrix(1,0,0,-1,-71.95,63.5)">
				<tspan style="font-size: 12.50px; " x="0" y="0">+</tspan>
			</text>

			<text transform="matrix(1,0,0,-1,-63.39,63.5)">
				<tspan style="font-size: 12.50px; " x="0" y="0">G</tspan>
			</text>

		</g>
	</g>
</svg>), codon 44 (<svg style="vertical-align:-0.17554pt;width:21.25px;" id="M8" height="10.9125" version="1.1" viewBox="0 0 21.25 10.9125" width="21.25"  xmlns="http://www.w3.org/2000/svg">
	<g transform="matrix(1.25,0,0,-1.25,0,10.9125)">
		<g transform="translate(72,-63.27)">
			<text transform="matrix(1,0,0,-1,-71.95,63.5)">
				<tspan style="font-size: 12.50px; " x="0" y="0">−</tspan>
			</text>

			<text transform="matrix(1,0,0,-1,-63.39,63.5)">
				<tspan style="font-size: 12.50px; " x="0" y="0">C</tspan>
			</text>

		</g>
	</g>
</svg>), and codon 5 (<svg style="vertical-align:-0.17554pt;width:30.799999px;" id="M9" height="10.9125" version="1.1" viewBox="0 0 30.799999 10.9125" width="30.799999"  xmlns="http://www.w3.org/2000/svg">
	<g transform="matrix(1.25,0,0,-1.25,0,10.9125)">
		<g transform="translate(72,-63.27)">
			<text transform="matrix(1,0,0,-1,-71.95,63.5)">
				<tspan style="font-size: 12.50px; " x="0" y="0">−</tspan>
			</text>

			<text transform="matrix(1,0,0,-1,-63.39,63.5)">
				<tspan style="font-size: 12.50px; " x="0" y="0">C</tspan>
				<tspan style="font-size: 12.50px; " x="8.3395014" y="0">T</tspan>
			</text>

		</g>
	</g>
</svg>). There were some notable differences in frequencies of various mutations in comparison to other Eastern Mediterranean populations, as well as between the two provinces studied. The latter illustrates the relative heterogeneity of the mutations distribution in Iraq, and the need to screen other areas of the country, to ensure establishing an effective prenatal program.

Nasir A. S. Al-Allawi

Kawa M. A. Hassan

Anwar K. Sheikha

Farida F. Nerweiy

Raji S. Dawood

Jaladet  Jubrael

<svg style="vertical-align:-2.79825pt" height="15.25" version="1.1" viewBox="0 0 9.5625 15.25" width="9.5625" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg"><g transform="matrix(.017,-0,0,-.017,.062,11.712)"><path id="x1D737" d="M54 -114l114 487q24 102 62.5 165t102.5 109q53 38 129 38q51 0 86 -33.5t35 -84.5q0 -57 -31.5 -105t-81.5 -68v-2q88 -14 88 -126q0 -64 -29 -127q-29 -62 -90 -107q-62 -44 -143 -44q-67 0 -97 18l-28 -124q-11 -51 -26 -87h-117q9 19 26 91zM304 480l-87 -393&#xA;q0 -29 19 -44t48 -15q63 0 110 74q46 74 46 153q0 117 -52 117q-6 0 -23.5 -3t-21.5 -3q-33 0 -33 25q0 15 15 25.5t41 10.5q8 0 30 -6q27 -5 28 -5q26 0 41 43t15 89q0 39 -17.5 62t-44.5 23q-46 0 -71 -35.5t-43 -117.5z" /></g></svg>-Thalassemia  Mutations among Transfusion-Dependent Thalassemia Major Patients in Northern Iraq

Heat shock protein 70 (HSP 70) plays a vital role by bestowing cytoprotection against diverse kinds of stresses. The ubiquitous HSP 70 proteins are the most abundant and temperature sensitive among all the HSPs. The present paper has characterized HSP70-1 cDNA in goat (<i>Capra hircus</i>). Total RNA isolated from goat peripheral blood mononuclear cells was reverse transcribed to cDNA that was used for amplification of HSP 70-1 gene.  PCR product (1926&#x2009;bp) was cloned in pGEM-T easy vector and sequenced. Sequence analysis revealed 1926-bp-long open reading frame of HSP 70-1 gene encoding 641 amino acids in goat, as reported in cattle. At nucleotide level, goat HSP 70-1 was found to be 96&#8211;99&#37; similar to that of sheep (partial), cattle, and buffalo whereas the similarity at amino acid level was 95&#8211;100&#37;. Nonsynonymous substitutions exceeding synonymous substitutions indicate the evolution of this protein through positive selection among domestic animals. Goat and sheep appear to have diverged from a common ancestor in phylogenetic analysis. Predicted protein structures of goat HSP 70 protein obtained from deduced amino acid sequence indicated that the functional amino acids involved in chaperoning through ATPase hydrolytic cycle and in uncoating of clathrin coated vesicles are highly conserved.

Nitin Gade

R. K. Mahapatra

Arvind Sonawane

V. K. Singh

Ramesh Doreswamy

Mohini Saini

Molecular Characterization of Heat Shock Protein 70-1 Gene of Goat (<i>Capra hircus</i>)

Allele frequencies of ten autosomal STR loci, D3S1358, vWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, TH01, and FGA were investigated in Chakma and Tripura tribal populations of Bangladesh. In both the populations, all loci were in Hardy-Weinberg equilibrium except for FGA locus in Chakma and D21S11 in Tripura. All the loci were highly polymorphic in Chakma population with an observed heterozygosity <svg style="vertical-align:-2.21957pt;width:31.3375px;" id="M1" height="13.45" version="1.1" viewBox="0 0 31.3375 13.45" width="31.3375"  xmlns="http://www.w3.org/2000/svg">
	<g transform="matrix(1.25,0,0,-1.25,0,13.45)">
		<g transform="translate(72,-61.24)">
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				<tspan style="font-size: 12.50px; " x="0" y="0">(</tspan>
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				<tspan style="font-size: 12.50px; " x="15.078618" y="0">𝑜</tspan>
				<tspan style="font-size: 12.50px; " x="20.804993" y="0">)</tspan>
			</text>

		</g>
	</g>
</svg> of <svg style="vertical-align:-0.30096pt;width:10.825px;" id="M2" height="8.8500004" version="1.1" viewBox="0 0 10.825 8.8500004" width="10.825"  xmlns="http://www.w3.org/2000/svg">
	<g transform="matrix(1.25,0,0,-1.25,0,8.85)">
		<g transform="translate(72,-64.92)">
			<text transform="matrix(1,0,0,-1,-71.95,65.27)">
				<tspan style="font-size: 12.50px; " x="0" y="0">&gt;</tspan>
			</text>

		</g>
	</g>
</svg>0.7 and moderately polymorphic in Tripura population <svg style="vertical-align:-2.21957pt;width:70.262497px;" id="M3" height="13.575" version="1.1" viewBox="0 0 70.262497 13.575" width="70.262497"  xmlns="http://www.w3.org/2000/svg">
	<g transform="matrix(1.25,0,0,-1.25,0,13.575)">
		<g transform="translate(72,-61.14)">
			<text transform="matrix(1,0,0,-1,-71.95,63.4)">
				<tspan style="font-size: 12.50px; " x="0" y="0">(</tspan>
				<tspan style="font-size: 12.50px; " x="4.1634989" y="0">𝐻</tspan>
				<tspan style="font-size: 12.50px; " x="15.078618" y="0">𝑜</tspan>
				<tspan style="font-size: 12.50px; " x="24.268324" y="0">&gt;</tspan>
				<tspan style="font-size: 12.50px; " x="36.308712" y="0">0</tspan>
				<tspan style="font-size: 12.50px; " x="42.560211" y="0">.</tspan>
				<tspan style="font-size: 12.50px; " x="45.685963" y="0">6</tspan>
				<tspan style="font-size: 12.50px; " x="51.937462" y="0">)</tspan>
			</text>

		</g>
	</g>
</svg>. However, both the population showed least polymorphism at TH01 locus <svg style="vertical-align:-2.21957pt;width:70.262497px;" id="M4" height="13.575" version="1.1" viewBox="0 0 70.262497 13.575" width="70.262497"  xmlns="http://www.w3.org/2000/svg">
	<g transform="matrix(1.25,0,0,-1.25,0,13.575)">
		<g transform="translate(72,-61.14)">
			<text transform="matrix(1,0,0,-1,-71.95,63.4)">
				<tspan style="font-size: 12.50px; " x="0" y="0">(</tspan>
				<tspan style="font-size: 12.50px; " x="4.1634989" y="0">𝐻</tspan>
				<tspan style="font-size: 12.50px; " x="15.078618" y="0">𝑜</tspan>
				<tspan style="font-size: 12.50px; " x="24.268324" y="0">&lt;</tspan>
				<tspan style="font-size: 12.50px; " x="36.308712" y="0">0</tspan>
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				<tspan style="font-size: 12.50px; " x="45.685963" y="0">6</tspan>
				<tspan style="font-size: 12.50px; " x="51.937462" y="0">)</tspan>
			</text>

		</g>
	</g>
</svg>. A comparison between Chakma and Tripura population data revealed statistically significant differences in allele frequency distribution for most of the loci. A similar comparison with the mainstream Bengali population using previously published data from this lab also showed significant difference in allele frequency with these two tribal populations.

Ahmad Ferdous

Mohammad Eunus Ali

Shafiul Alam

Mahamud Hasan

Tania Hossain

Sharif Akhteruzzaman

Allele Frequencies of 10 Autosomal STR Loci from Chakma and Tripura Tribal Populations in Bangladesh

TLR9 plays pivotal role in innate immune responses through upregulation of costimulatory molecules and induction of proinflammatory cytokines like type I interferons including interferon alpha (IFNA). The present study characterized IFNA cDNA and predicted protein sequences in goat and black buck. Response of the PBM cells to TLR9 agonist CpG ODN C and Phorbol Myristate Acetate (PMA) was evaluated by realtime PCR. IFNA coding sequences were amplified from leukocyte cDNA and cloned in pGEMT-easy vector for nucleotide sequencing. Sequence analysis revealed 570 bp, IFNA ORF encoding 189 amino acids in goat and black buck. Black buck and goat IFNA has 92.1% to 94.7% and 93% to 95.6% similarity at nucleotide level, 86.3% to 89.5% and 70.9% to 91.6% identity at amino acid level with other ruminants, respectively. Nonsynonymous substitutions exceeding synonymous substitutions indicated IFNA evolved through positive selection among ruminants. In spite of lower total leukocyte count, the innate immune cells like monocytes and neutrophils were more in black buck compared to goat. In addition, CpG ODN C-stimulated PBM cells revealed raised IFNA transcript in black buck than goat. These findings indicate sturdy genetically governed immune system in wild antelope black buck compared to domestic ruminant goat.

Devendra Swarup

Vivek Kumar Singh

Suchitra Upreti

Asit Das

Praveen K. Gupta

Interferon Alpha Characterization and Its Comparative Expression in PBM Cells of <i>Capra hircus</i> and <i>Antelope cervicapra</i> Cultured in the Presence of TLR9 Agonist

<i>Mycobacterium avium</i> subsp. <i>paratuberculosis</i> (Map) contains PPE family antigens which are Proline and glutamic acid rich and may play important role as T cell antigens. Hence the identification and generation of antigens are necessary for immunological characterization. In the present study, the epitopic region of a unique PPE gene encoding 34.9&#x2009;kDa protein from Map was amplified by polymerase chain reaction. The gene was cloned into <i>Escherichia coli</i> vector pQE30 UA. The recombinant plasmid designated as pQPPE was transformed into <i>E. coli</i> M15 and induced with IPTG revealed the high level expression of 37.1 kDa His-fusion protein (34.9&#x2009;kDa PPE and 2.2&#x2009;kDa His-tag), which was confirmed by immunoblotting. Recombinant PPE protein was then purified by Ni-NTA agarose chromatography. The polyclonal antiserum raised against purified recombinant PPE protein reacted with expressed 37.1&#x2009;kDa His-fusion protein as well as with Map sonicate. The protein elicited significant delayed type hypersensitivity (DTH) skin reaction in mice sensitized with Map. The results indicated that the recombinant PPE protein of Map was associated with cellular immune response.

Rajib Deb

P. P. Goswami

Expression of a Gene Encoding 34.9&#x2009;kDa PPE Antigen of <i>Mycobacterium avium</i> subsp. <i>paratuberculosis</i> in <i>E. coli</i>

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Molecular Biology International

Table of Contents

Table of Contents: 2010

Tyrosinase Small Interfering RNA Effectively Suppresses Tyrosinase Gene Expression In Vitro and In Vivo

Tissue-Specific Expression of the Chicken Calpain2 Gene

-Thalassemia Mutations among Transfusion-Dependent Thalassemia Major Patients in Northern Iraq

Molecular Characterization of Heat Shock Protein 70-1 Gene of Goat (Capra hircus)

Allele Frequencies of 10 Autosomal STR Loci from Chakma and Tripura Tribal Populations in Bangladesh

Interferon Alpha Characterization and Its Comparative Expression in PBM Cells of Capra hircus and Antelope cervicapra Cultured in the Presence of TLR9 Agonist

Expression of a Gene Encoding 34.9 kDa PPE Antigen of Mycobacterium avium subsp. paratuberculosis in E. coli