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The Use of Various PCR Strategies to Identify a Bovine Relaxin or Relaxin-like Gene Product

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The Use of Various PCR Strategies to Identify a Bovine Relaxin or Relaxin-like Gene Product
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Thompson, Anna
Fields, Michael
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Journal of Undergraduate Research
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..Oluliie 6, issue 1 - Septiinel: r 21:11:14



The Use of Various PCR Strategies to Identify a Bovine Retaxin or Retaxin-
like Gene Product


Anna Thompson


ABSTRACT


In most mammalian species, the structure of relaxin has been determined and the sites of production have

been characterized through immunoreactivity and mRNA localization studies. However, in the cow, there has yet

to be a clear identification of a hormone with a relaxin-like structure, despite numerous studies conducted on

the subject. The cow will respond to exogenous administration of relaxin, which would seem to indicate a

physiology that is similar to that of non-ruminant species. The purpose of this experiment was to attempt to

identify a bovine relaxin gene or relaxin pseudogene through the use of the polymerase chain reaction (PCR).

The primers used for these PCR studies were based on previous published works concerning the presence of a

relaxin pseudogene in the sheep and the recently discovered relaxin 3 gene in the pons/medulla tissue of the

human and the mouse. These PCR procedures did not result in any PCR products isolated from cow tissues.

This indicates that the cow does not contain a similar relaxin pseudogene to the one found in the sheep. In

addition, these results indicate that the cow does not have a relaxin 3 gene that corresponds to a novel bovine

EST-clone expressed in the pons/medulla tissue of the brain. Future studies will need to be conducted on other

parts of the brain to investigate the possibility that relaxin 3 expression is localized elsewhere.



INTRODUCTION


Relaxin is a polypeptide hormone consisting of two polypeptide chains that are synthesized as a B-chain/C-peptide/

A-chain precursor. It is secreted in the highest amounts by the ovary during pregnancy and has a number of roles

in mammals that are generally associated with the female reproductive tract (Bathgate et al., 2002). Some of

these roles include the inhibition of uterine activity as well as the relaxation of the pubic symphysis and dilation of

the cervix prior to parturition. Other biological effects of relaxin may be associated with the male reproductive

system and a possible role in the central nervous system (Roche et al., 1993).



In most mammalian species, the structure of relaxin has been determined and the sites of production have

been characterized through immunoreactivity and mRNA localization studies (Sherwood, 1988). However,

in ruminants, there has yet to be a clear identification of a hormone with a relaxin-like structure (Ivell et al.,

1994). The cow in particular has been shown to possess normal relaxin physiology and will respond to

exogenous administration of porcine relaxin, which would seem to indicate a physiology similar to that of






non-ruminant species (Perezgrovas and Anderson, 1982). Recently, Roche et al. reported a relaxin-like

sequence from the ewe, which has all of the characteristics of a pseudogene (1993). This sequence includes a

short open reading frame which only corresponds to a segment of the relaxin A-chain, has numerous stop

codons, and is not represented as a functional mRNA in any reproductive tissues of the sheep.



The purpose of this study was to attempt to identify a relaxin or relaxin-like gene sequence in the cow.

The characterization of relaxin in the cow is important because of its potential clinical applications in the field

of veterinary medicine as well as its use as an experimental model for investigating relaxin physiology. In order

to achieve these objectives, a PCR study was conducted using bovine placental tissues and the primers identified

by Roche et al. as characterizing a relaxin pseudogene. In addition, a PCR study was conducted on bovine

pons/medulla tissue to search for the recently discovered relaxin 3 gene. (Bathgate et al., 2002). This gene has

been successfully characterized in the pons/medulla tissue of the human and the mouse, and has sequence

homology with a novel Bovine relaxin-3 clone isolated from the expressed sequence tag (EST) database

(Bathgate and Ivell, September 2, 2003, personal communication).



MATERIALS AND METHODS


RNA Isolation


Total cellular RNA was isolated from chorio-allantois (placental) tissue collected from late pregnant ewes and

cows. Total RNA was also isolated from brain tissue from the mouse (positive control) and pons/medulla brain

tissue from the cow. Sheep and mice tissues were collected from the UF Meat Processing Center and from the

UF Medical School, respectively. Cow tissue was collected from Central Beef processing plant located in Center

Hill, Florida. Total RNA was extracted according to protocol established by Life Technologies utilizing 3 ug of

tissue and Trizol reagant. Upon completion of the extraction procedure, absorbance readings were measured using

a spectrophotometer to ensure that the RNA was of adequate quality and was not contaminated.



PCR Amplification


Total RNA was reverse transcribed according to protocol provided with the cDNA Cycle Kit of Invitrogen

Life Technologies. The reverse transcription reaction contained 5 pg of total cellular RNA, 1 pL of oligo DT primer,

1 pL of RNase inhibitor, 4 pL of 5X RT buffer, 1 pL of 100mM dNTPs, 1 pL of 80mM sodium pyrophosphate, and

0.5 pL of AMV reverse transcriptase that were reacted at 42oC for 1 hour. Upon denaturation of the RNA-

cDNA hybrids at 95oC for two minutes, a 2 pL aliquot of cDNA was added to a 50 pL PCR reaction containing 5 pL

of 10X PCR buffer, 1 pL 100mM dNTPs, 0.3 pg of specific primers, and 1 unit of taq polymerase and was reacted

for 40 cycles (94oC for 1 minute, 55oC for 2 minutes, and 72oC for 3 minutes) in a thermal cycler. The PCR

products where then analyzed on a 1.5% agarose gel.



Chorio-allantois (placental) tissue from late pregnant ewes and cows were screened with specific forward





(5'-GGCAGCCATCATTGAGAGAGCAACTA-3') and reverse (5'-GCACAGCTTCATTTCAGCACAATTGAA-3')

primers corresponding to the primers used by Roche et al. as characterizing a pseudogene in the sheep. Mouse

brain tissue was screened with specific forward (5'-TGCGGAGGCTCACGATGGCGC-3') and reverse

(5'-GACAGCAGCTTGCAGGCACGG-3') primers corresponding to primers used by Bathgate et al. as characterizing

the M3 relaxin gene. Cow pons/medulla tissue was screened using specific forward

(5'-GGACATGCTGGCCCATGAAGC-3') and reverse (5'-TGGAGAACCAGAGTGAATGTC-3') primers that correspond to

a novel Bovine relaxin-3 EST clone.



RESULTS


Characterization of a Bovine Relaxin or Relaxin-like Gene Product


PCR was performed on chorio-allantois (placental) tissue from late pregnant ewes and cows using specific

primers characterized as corresponding to a sheep relaxin pseudogene (Roche et al., 1993). The results of the

PCR indicated the presence of a PCR product in the sheep, which was consistent with previously established

results. No PCR products were obtained from cow chorio-allantois tissue (Figure 1). The PCR protocol was

repeated using different PCR buffers but no additional PCR products were obtained from this variation.




L 1 2 3 4









Figure 1. Analysis of PCR products on 1.5% agarose gel: lane L, ladder; lane 1, positive control; lane 2-

4, cow chorio-allantois tissue.Characterization of a Bovine Relaxin 3 Gene Product.







PCR was first performed on mouse brain tissue using specific primers characterized as being associated with the

M3 relaxin gene to serve as control for work on the cow (Bathgate et al., 2002). The results from the mouse

study indicated the presence of a PCR product, which was consistent with previously published works (Bathgate

et al., 2002). Cow pons/medulla tissue was then screened using specific primers designed as corresponding to

a novel Bovine relaxin-3 EST clone. No PCR products were isolated from cow pons/medulla tissue (Figure 2). The

PCR protocol was repeated using different PCR buffers but no additional PCR products were obtained.






















Figure 2. Analysis of PCR products on 1.5% agarose gel: lane L, ladder; lane 1, positive control; lane

2, cow pons/medulla tissue.




DISCUSSION


The cow has been shown to exhibit a normal relaxin physiology in response to exogenous administration of

relaxin, which would seem to indicate a relaxin physiology that is similar to non-ruminant species (Perezgrovas

and Anderson, 1982). However, no significant evidence to date has shown that the cow has the capability

of synthesizing a relaxin or relaxin-like gene product, even though extensive studies have been conducted on

this subject (Ivell et al., 1994). In the first part of this experiment, we attempted to isolate such a gene

product through a series of PCR procedures using a set of primers identified as characterizing a relaxin pseudogene

in the sheep (Roche et al., 1993). Since the sheep is a close ruminant relative of the cow, it is reasonable to

suspect that the cow might also have a similar pseudogene. However, the lack of any PCR products in

this experiment suggests that the cow does not possess a similar pseudogene to the one found in the sheep.

This result would seem to indicate the possibility that the cow evolved after the sheep, and simply lost

the pseudogene while retaining the ability to respond to the gene product by conserving the gene

receptor. Phylogenetic studies on ruminant evolution seem to support this notion (Scott and Janis, 1993).

Another possibility is that the cow relaxin gene may have such low homology as compared with other species

that PCR is ineffective in isolating any gene products.



Relaxin 3 is a novel relaxin gene that has recently been identified in both the human (H3) and mouse (M3)

genome (Bathgate et al., 2002). The highest level of expression of this gene is in the brain, which supports the

idea that relaxin has many functional roles outside of the female reproductive tract. This gene has also been found

to retain the structural features necessary for the activation of relaxin receptors (Bathgate et al., 2002).

This evidence could possibly explain why the cow would lack the actual relaxin gene and still retain the

relaxin receptors necessary to respond to exogenous administration of relaxin. In the second half of our

experiment, we attempted to isolate a bovine relaxin 3 gene from tissue isolated from the pons/medulla of the

cow through a series of PCR procedures using primers designed from a novel bovine 3 EST clone. The

preliminary results of this study have not yet isolated any PCR products. However, it is possible that the

bovine relaxin 3 expression may be localized in other areas of the brain besides that of the pons/medulla.





This hypothesis will be tested in future studies conducted by our laboratory.


REFERENCES


1. Bathgate, R., Samuel, C., Burazin, T., Layfield, S., Claasz, A., Reytomas, I., Dawson, N., Zhao, C., Bond,

C., Summers, R., Parry, L., Wade, J., and Tregear, G. (2002). Human Relaxin Gene 3 (H3) and the Equivalent

Mouse Relaxin (M3) Gene. Journal of Biological Chemistry 277(2), 1148-1157.

2. Ivell, R., Hartung, S., Abend, N., Rust, W., and Hunt, N. (1994). The Search for Bovine Relaxin. Progress in

Relaxin Research: Proceedings of the 2nd International Conference on the Hormone Relaxin. 439-456.

3. Perezgrovas, R. and Anderson, L. (1982). Effect of Porcine Relaxin on Cervical Dilation, Pelvic Area and Parturition

in Beef Heifers. Biology of Reproduction, 26-, 765-776.

4. Roche, P., Crawford, R., and Tregear, G. (1993). A Single-copy Relaxin-like Gene Sequence is Present in

Sheep. Molecular and Cellular Endocrinology, 91, 21-28.

5. Scott, K. and Janis, C. (1993). Relationships of the Ruminantia and an analysis of the characters used in

ruminant taxonomy. Mammalian Phylogeny, (F. Szaly., M. Novacek., and M. McKenna., eds.), Springer-Verlag,

New York, 282-302.

6. Sherwood, O.D. (1988). The Physiology of Reproduction (E. Knobil. and J. .Neil, eds.), Raven Press, New York,

585-673. The paper will be 2,000 words long.


Back to the Journal of Undergraduate Research


College of Liberal Arts and Sciences I University Scholars Program I University of Florida I


� University of Florida, Gainesville, FL 32611; (352) 846-2032.


UP UNIVERSITY of
SFLORIDA
,WSmnfls :rft (nilt N' F n




Full Text

PAGE 1

Journal of Undergraduate Research Volume 6, Issue 1 September 2004The Use of Various PCR Strategies to Identify a Bovine Relaxin or Relaxinlike Gene Product Anna Thompson ABSTRACTIn most mammalian species, the structure of relaxin has been determined and the sites of production have been characterized through immunoreactivity and mRNA localization studies. However, in the cow, there has yet to be a clear identification of a hormone with a relaxin-like structure, despite numerous studies conducted on the subject. The cow will respond to exogenous administration of relaxin, which would seem to indicate a physiology that is similar to that of non-ruminant species. The purpose of this experiment was to attempt to identify a bovine relaxin gene or relaxin pseudogene through the use of the polymerase chain reaction (PCR). The primers used for these PCR studies were based on previous published works concerning the presence of a relaxin pseudogene in the sheep and the recently discovered relaxin 3 gene in the pons/medulla tissue of the human and the mouse. These PCR procedures did not result in any PCR products isolated from cow tissues. This indicates that the cow does not contain a similar relaxin pseudogene to the one found in the sheep. In addition, these results indicate that the cow does not have a relaxin 3 gene that corresponds to a novel bovine EST-clone expressed in the pons/medulla tissue of the brain. Future studies will need to be conducted on other parts of the brain to investigate the possibility that relaxin 3 expression is localized elsewhere.INTRODUCTIONRelaxin is a polypeptide hormone consisting of two polypeptide chains that are synthesized as a B-chain/C-peptide/ A-chain precursor. It is secreted in the highest amounts by the ovary during pregnancy and has a number of roles in mammals that are generally associated with the female reproductive tract (Bathgate et al., 2002). Some of these roles include the inhibition of uterine activity as well as the relaxation of the pubic symphysis and dilation of the cervix prior to parturition. Other biological effects of relaxin may be associated with the male reproductive system and a possible role in the central nervous system (Roche et al., 1993). In most mammalian species, the structure of relaxin has been determined and the sites of production have been characterized through immunoreactivity and mRNA localization studies (Sherwood, 1988). However, in ruminants, there has yet to be a clear identification of a hormone with a relaxin-like structure (Ivell et al., 1994). The cow in particular has been shown to possess normal relaxin physiology and will respond to exogenous administration of porcine relaxin, which would seem to indicate a physiology similar to that of

PAGE 2

non-ruminant species (Perezgrovas and Anderson, 1982). Recently, Roche et al. reported a relaxin-like sequence from the ewe, which has all of the characteristics of a pseudogene (1993). This sequence includes a short open reading frame which only corresponds to a segment of the relaxin A-chain, has numerous stop codons, and is not represented as a functional mRNA in any reproductive tissues of the sheep. The purpose of this study was to attempt to identify a relaxin or relaxin-like gene sequence in the cow. The characterization of relaxin in the cow is important because of its potential clinical applications in the field of veterinary medicine as well as its use as an experimental model for investigating relaxin physiology. In order to achieve these objectives, a PCR study was conducted using bovine placental tissues and the primers identified by Roche et al. as characterizing a relaxin pseudogene. In addition, a PCR study was conducted on bovine pons/medulla tissue to search for the recently discovered relaxin 3 gene. (Bathgate et al., 2002). This gene has been successfully characterized in the pons/medulla tissue of the human and the mouse, and has sequence homology with a novel Bovine relaxin-3 clone isolated from the expressed sequence tag (EST) database (Bathgate and Ivell, September 2, 2003, personal communication). MATERIALS AND METHODSRNA Isolation Total cellular RNA was isolated from chorio-allantois (placental) tissue collected from late pregnant ewes and cows. Total RNA was also isolated from brain tissue from the mouse (positive control) and pons/medulla brain tissue from the cow. Sheep and mice tissues were collected from the UF Meat Processing Center and from the UF Medical School, respectively. Cow tissue was collected from Central Beef processing plant located in Center Hill, Florida. Total RNA was extracted according to protocol established by Life Technologies utilizing 3 ug of tissue and Trizol reagant. Upon completion of the extraction procedure, absorbance readings were measured using a spectrophotometer to ensure that the RNA was of adequate quality and was not contaminated. PCR Amplification Total RNA was reverse transcribed according to protocol provided with the cDNA Cycle Kit of Invitrogen Life Technologies. The reverse transcription reaction contained 5 g of total cellular RNA, 1 L of oligo DT primer, 1 L of RNase inhibitor, 4 L of 5X RT buffer, 1 L of 100mM dNTPs, 1 L of 80mM sodium pyrophosphate, and 0.5 L of AMV reverse transcriptase that were reacted at 42oC for 1 hour. Upon denaturation of the RNAcDNA hybrids at 95oC for two minutes, a 2 L aliquot of cDNA was added to a 50 L PCR reaction containing 5 L of 10X PCR buffer, 1 L 100mM dNTPs, 0.3 g of specific primers, and 1 unit of taq polymerase and was reacted for 40 cycles (94oC for 1 minute, 55oC for 2 minutes, and 72oC for 3 minutes) in a thermal cycler. The PCR products where then analyzed on a 1.5% agarose gel. Chorio-allantois (placental) tissue from late pregnant ewes and cows were screened with specific forward

PAGE 3

(5'-GGCAGCCATCATTGAGAGAGCAACTA-3') and reverse (5'-GCACAGCTTCATTTCAGCACAATTGAA-3') primers corresponding to the primers used by Roche et al. as characterizing a pseudogene in the sheep. Mouse brain tissue was screened with specific forward (5'-TGCGGAGGCTCACGATGGCGC-3') and reverse (5'-GACAGCAGCTTGCAGGCACGG-3') primers corresponding to primers used by Bathgate et al. as characterizing the M3 relaxin gene. Cow pons/medulla tissue was screened using specific forward (5'-GGACATGCTGGCCCATGAAGC-3') and reverse (5'-TGGAGAACCAGAGTGAATGTC-3') primers that correspond to a novel Bovine relaxin-3 EST clone.RESULTSCharacterization of a Bovine Relaxin or Relaxin-like Gene Product PCR was performed on chorio-allantois (placental) tissue from late pregnant ewes and cows using specific primers characterized as corresponding to a sheep relaxin pseudogene (Roche et al., 1993). The results of the PCR indicated the presence of a PCR product in the sheep, which was consistent with previously established results. No PCR products were obtained from cow chorio-allantois tissue (Figure 1). The PCR protocol was repeated using different PCR buffers but no additional PCR products were obtained from this variation. Figure 1. Analysis of PCR products on 1.5% agarose gel: lane L, ladder; lane 1, positive control; lane 24, cow chorio-allantois tissue.Characterization of a Bovine Relaxin 3 Gene Product. PCR was first performed on mouse brain tissue using specific primers characterized as being associated with the M3 relaxin gene to serve as control for work on the cow (Bathgate et al., 2002). The results from the mouse study indicated the presence of a PCR product, which was consistent with previously published works (Bathgate et al., 2002). Cow pons/medulla tissue was then screened using specific primers designed as corresponding to a novel Bovine relaxin-3 EST clone. No PCR products were isolated from cow pons/medulla tissue (Figure 2). The PCR protocol was repeated using different PCR buffers but no additional PCR products were obtained.

PAGE 4

Figure 2. Analysis of PCR products on 1.5% agarose gel: lane L, ladder; lane 1, positive control; lane 2, cow pons/medulla tissue.DISCUSSIONThe cow has been shown to exhibit a normal relaxin physiology in response to exogenous administration of relaxin, which would seem to indicate a relaxin physiology that is similar to non-ruminant species (Perezgrovas and Anderson, 1982). However, no significant evidence to date has shown that the cow has the capability of synthesizing a relaxin or relaxin-like gene product, even though extensive studies have been conducted on this subject (Ivell et al., 1994). In the first part of this experiment, we attempted to isolate such a gene product through a series of PCR procedures using a set of primers identified as characterizing a relaxin pseudogene in the sheep (Roche et al., 1993). Since the sheep is a close ruminant relative of the cow, it is reasonable to suspect that the cow might also have a similar pseudogene. However, the lack of any PCR products in this experiment suggests that the cow does not possess a similar pseudogene to the one found in the sheep. This result would seem to indicate the possibility that the cow evolved after the sheep, and simply lost the pseudogene while retaining the ability to respond to the gene product by conserving the gene receptor. Phylogenetic studies on ruminant evolution seem to support this notion (Scott and Janis, 1993). Another possibility is that the cow relaxin gene may have such low homology as compared with other species that PCR is ineffective in isolating any gene products. Relaxin 3 is a novel relaxin gene that has recently been identified in both the human (H3) and mouse (M3) genome (Bathgate et al., 2002). The highest level of expression of this gene is in the brain, which supports the idea that relaxin has many functional roles outside of the female reproductive tract. This gene has also been found to retain the structural features necessary for the activation of relaxin receptors (Bathgate et al., 2002). This evidence could possibly explain why the cow would lack the actual relaxin gene and still retain the relaxin receptors necessary to respond to exogenous administration of relaxin. In the second half of our experiment, we attempted to isolate a bovine relaxin 3 gene from tissue isolated from the pons/medulla of the cow through a series of PCR procedures using primers designed from a novel bovine 3 EST clone. The preliminary results of this study have not yet isolated any PCR products. However, it is possible that the bovine relaxin 3 expression may be localized in other areas of the brain besides that of the pons/medulla.

PAGE 5

This hypothesis will be tested in future studies conducted by our laboratory. REFERENCES1. Bathgate, R., Samuel, C., Burazin, T., Layfield, S., Claasz, A., Reytomas, I., Dawson, N., Zhao, C., Bond, C., Summers, R., Parry, L., Wade, J., and Tregear, G. (2002). Human Relaxin Gene 3 (H3) and the Equivalent Mouse Relaxin (M3) Gene. Journal of Biological Chemistry 277(2), 1148-1157. 2. Ivell, R., Hartung, S., Abend, N., Rust, W., and Hunt, N. (1994). The Search for Bovine Relaxin. Progress in Relaxin Research: Proceedings of the 2nd International Conference on the Hormone Relaxin. 439-456. 3. Perezgrovas, R. and Anderson, L. (1982). Effect of Porcine Relaxin on Cervical Dilation, Pelvic Area and Parturition in Beef Heifers. Biology of Reproduction, 26-, 765-776. 4. Roche, P., Crawford, R., and Tregear, G. (1993). A Single-copy Relaxin-like Gene Sequence is Present in Sheep. Molecular and Cellular Endocrinology, 91, 21-28. 5. Scott, K. and Janis, C. (1993). Relationships of the Ruminantia and an analysis of the characters used in ruminant taxonomy. Mammalian Phylogeny, (F. Szaly., M. Novacek., and M. McKenna., eds.), Springer-Verlag, New York, 282-302. 6. Sherwood, O.D. (1988). The Physiology of Reproduction (E. Knobil. and J. .Neil, eds.), Raven Press, New York, 585-673. The paper will be 2,000 words long. Back to the Journal of Undergraduate Research College of Liberal Arts and Sciences | University Scholars Program | University of Florida | University of Florida, Gainesville, FL 32611; (352) 846-2032.


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