Patent Application: US-52291608-A

Abstract:
methods and materials for identifying bovine males which will produce semen which will exhibit a higher rate of successfully impregnation by either artificial insemination or natural mating . the method employs snps that have been identified in the bdazl gene which are associated with enhanced male fertility in bovine males and haplotypes formed from such snps . the method herein can be used to identify male dairy or beef cattle . the method herein can be applied to bovine animals at an appropriate time and particularly at birth or in utero . the invention further provides kits for conduction assays to assess bovine male fertility / infertility . the invention additionally provides a method of breeding cattle employing the methods and materials herein .

Description:
a description of the cloning and sequence analysis of bdazl is provided in the examples . the sequences of the full - length cdna clone and the predicted amino acid sequence of the bdazl gene is provided in fig4 . additional summary results are provided in fig5 , 6 and 7 . the following was determined from this analysis of bdazl . there is no daz gene on the bovine y chromosome , but there is a dazl gene on the autosome in the bovine genome . the full - length bdazl cdna is predicted to encode a protein of 295 amino acids with an rrm . the deduced protein sequence of bdazl is 96 % similar to human dazl and 97 % similar to mouse dazl . fluorescence in situ hybridization ( fish ) maps bdazl to the distal region on bta1q . the bdazl consists of 11 exons and 10 introns , spanning ˜ 33 kb in the genome . a bdazl pseudogene was identified on bta16 . expression analysis of bdazl in 13 different tissues by rt - pcr shows that two transcripts , variant 1 and 2 , of the bdazl gene are detected only in testis mrna . the transcript variants are believed to result from alternative rna splicing , because variant 1 contains an additional 1623 bp insertion in the 3 ′ utr . a sequence motif “ guuc ” that can activate the translation of its own mrna is present in the 3 ′ utr in bdazl variant 2 , while four additional “ guuc ” motifs are present in variant 1 within the 1623 bp region . these results provided the groundwork for snp discovery and functional studies of the bdazl gene in cattle . two different approaches were applied in this research . the first approach was an in silico bioinformatics approach . we searched the bovine expressed sequence tags “ ests ” databases available online at tigr and genbank using the bdazl cdna sequence and identified two ests related to bdazl . these ests were compared by sequence alignment . only one nucleotide substitution ( c → g ) at nt 2771 of the bdazl cdna was identified and was considered as a potential snp . because there were not enough bdazl - related ests in the databases , a second approach was applied by pooling dna samples from fertile bulls as well as some subfertile bulls from one of the world &# 39 ; s largest ai companies ( the semex alliance , canada ) to identify snps . the fertile bulls included the top 10 and bottom 10 bulls as assessed by the provided , at least in part by non - return rate , which is a measure of the success of an insemination event . dna samples pooled from the top 10 , bottom 10 bulls , and 4 subfertile bulls were used in pcr ( table 1 ) and then the pcr fragments were sequenced . in addition , we have collected a dna sample from an infertile bull , which was also added to the pooled dna templates ( we referred to this as the master pooled dna ). as both bdazl transcripts ( 2996 bp and 1383 bp ) are large , spanning ˜ 33 kb in the genome , the discovery of snps was initially focused on all exons and the 3 ′ utr region that contains the sequence “ guuc ” motifs . bdazl snps : by the pooled - dna - pcr - sequencing approach , we have identified 16 snps ( table 2a ). among all exons , only one snp was identified from the exon 10 , nt 953 , with a silent t → c substitution . at the protein level , this snp does not change the serine amino acid . six snps were identified from the 3 ′ utr region , and five snps were identified from the intron sequences adjacent to the exons . once a snp is identified , it can be verified by two methods , one is pcr - rflp method , the other , pcr - sequencing . for the pcr - rflp , genomic dna from animals with different genotypes for the snp is used in pcr . the amplified products are subjected to restriction digestion with an enzyme recognizing the mutation site ( snp ). for the pcr - sequencing method , we run pcr using three sub - pooled dna templates , the top 10 , the bottom 10 , and the subfertile . the purified pcr fragments were sequenced at nevada genomic center ( ngc ). the sequences were compared to the original cdna sequence and the sequence obtained from the master pool to validate the snps . those snps that have been confirmed can be used in association studies . table 2b provides specific locations of snps in bdazl introns . snps were identified in intronic regions adjacent and either upstream or downstream of exons . more specifically snps associated with male fertility are found in intronic regions within about 300 bp upstream or downstream of exons of the dazl gene . 10 snps among 25 bulls ( top 10 , bottom 10 , 4 subfertile and 1 infertile ) ( table 3 ) have been genotyed . the genotype results are listed in table 4 . table 5 genotype and haplotype analysis probability individual of gametic id genotype parental haplotype 1 parental haplotype 2 assignment qfert * 1 a - g - a - g - g - t - a - a - c - t / a - g - a - g - g - t - a - a - c - t g - g - a - g - g - t - a - a - g - t 1 74 g - g - a - g - g - t - a - a - g - t 2 a - a - c - g - g - c - t - a - c - c / a - a - c - g - g - c - t - a - c - c a - a - c - g - g - c - t - a - c - c 1 73 a - a - c - g - g - c - t - a - c - c 3 a - g - a - g - g - t - a - g - c - t / a - g - a - g - g - t - a - g - c - t g - g - a - g - g - t - a - a - g - t 1 73 g - g - a - g - g - t - a - a - g - t 4 a - a - c - g - g - c - t - a - c - c / a - a - c - g - g - c - t - a - c - c a - g - a - g - g - t - a - a - c - t 1 73 a - g - a - g - g - t - a - a - c - t 5 a - g - a - g - a - t - a - g - c - t / a - g - a - g - a - t - a - g - c - t a - g - a - g - g - t - a - a - c - t 0 . 997352001 72 a - g - a - g - g - t - a - a - c - t 6 a - g - a - g - a - t - a - g - c - t / a - g - a - g - a - t - a - g - c - t g - g - a - g - g - t - a - a - g - t 0 . 661063159 72 g - g - a - g - g - t - a - a - g - t 7 a - a - c - g - g - c - t - a - c - c / a - a - c - g - g - c - t - a - c - c a - g - a - g - g - t - a - a - c - t 1 72 a - g - a - g - g - t - a - a - c - t 8 a - g - a - a - g - t - a - a - c - t / a - g - a - a - g - t - a - a - c - t a - g - a - g - g - t - a - a - c - t 1 72 a - g - a - g - g - t - a - a - c - t 9 a - a - c - g - g - c - t - a - c - c / a - a - c - g - g - c - t - a - c - c a - g - a - g - g - t - a - a - c - t 1 72 a - g - a - g - g - t - a - a - c - t 10 a - g - a - a - g - t - a - a - c - t / a - g - a - a - g - t - a - a - c - t a - g - a - g - a - t - a - g - c - t 1 72 a - g - a - g - a - t - a - g - c - t 11 a - g - a - g - g - t - a - a - c - t / a - g - a - g - g - t - a - a - c - t a - g - a - g - g - t - a - g - c - t 1 57 a - g - a - g - g - t - a - g - c - t 12 a - a - c - g - a - c - t - a - c - c / a - a - c - g - a - c - t - a - c - c a - g - a - g - g - t - a - a - c - t 0 . 986378256 58 a - g - a - g - g - t - a - a - c - t 13 a - g - a - a - g - t - a - a - c - t / a - g - a - a - g - t - a - a - c - t a - g - a - g - g - t - a - a - c - t 1 59 a - g - a - g - g - t - a - a - c - t 14 a - g - a - g - a - t - a - g - c - t / a - g - a - g - a - t - a - g - c - t g - g - a - g - g - t - a - a - g - t 1 59 g - g - a - g - g - t - a - a - g - t 15 a - g - a - g - g - t - a - a - c - t / a - g - a - g - g - t - a - a - c - t g - g - a - g - g - t - a - a - g - t 1 60 g - g - a - g - g - t - a - a - g - t 16 a - g - a - a - g - t - a - a - c - t / a - g - a - a - g - t - a - a - c - t g - g - a - g - g - t - a - a - g - t 1 60 g - g - a - g - g - t - a - a - g - t 17 g - g - a - g - g - t - a - a - g - t / g - g - a - g - g - t - a - a - g - t g - g - a - g - g - t - a - a - g - t 1 60 g - g - a - g - g - t - a - a - g - t 18 a - a - c - g - a - t - a - a - c - t / a - a - c - g - a - t - a - a - c - t a - g - a - g - g - t - a - a - c - t 0 . 978669963 61 a - g - a - g - g - t - a - a - c - t 19 a - a - c - g - g - c - t - a - c - c / a - a - c - g - g - c - t - a - c - c g - g - a - g - g - t - a - a - g - t 1 61 g - g - a - g - g - t - a - a - g - t 20 a - a - c - g - g - c - t - a - c - c / a - a - c - g - g - c - t - a - c - c a - g - a - g - g - t - a - a - c - t 1 62 a - g - a - g - g - t - a - a - c - t 21 a - a - c - g - a - t - a - a - c - t / a - a - c - g - a - t - a - a - c - t a - g - a - a - g - t - a - a - c - t 0 . 953114981 50 a - g - a - a - g - t - a - a - c - t 22 a - g - a - a - g - t - a - a - c - t / a - g - a - a - g - t - a - a - c - t a - g - a - g - g - t - a - g - c - t 0 . 999999911 54 a - g - a - g - g - t - a - g - c - t 23 a - g - a - g - g - t - a - a - c - t / a - g - a - g - g - t - a - a - c - t g - g - a - g - g - t - a - a - g - t 1 58 g - g - a - g - g - t - a - a - g - t 24 a - a - c - g - g - c - t - a - c - c / a - a - c - g - g - c - t - a - c - c a - g - a - g - a - t - a - g - c - t 0 . 838745952 59 a - g - a - g - a - t - a - g - c - t 25 a - a - c - g - a - c - t - a - c - c / a - a - c - g - a - c - t - a - c - c g - g - a - g - g - t - a - a - g - t 1 0 g - g - a - g - g - t - a - a - g - t * qfert stands for quantitative measurement of fertility ; the values listed here are the non - return rate ( nrr ). genotype and haplotype analysis . the sas / jmp software was used to analyze the genotype , haplotype and trait association of the 25 animals studied . table 5 shows the genotype and the most probable gametic assignments , along with nrr ( non - return rate ). binary haplotype association analysis —- high nrr vs subfertile ( sf ) group . the linkage disequilibrium ( ld ) test for allelic association between the high nrr group and subfertile group indicates that there is a very significant ld among the 10 snps and their alleles ( p & lt ; 0 . 0001 ) ( table 6 ). the test for marker - trait association also reveals a significant link between the snps and the fertility trait ( p & lt ; 0 . 005 ). further tests for haplotype - trait association have identified one haplotype ( a - a - c - a - a - t - a - a - c - t ) that is strongly associated to subfertile animals ( table 6 ) ( p & lt ; 0 . 001 ). binary haplotype association analysis — low nrr vs subfertile group . similar analysis indicates that there is no significant allelic association between low nrr and subfertile groups . this seems to be reasonable as the nrr overlaps between the low nrr ( range from 57 to 62 ) and the subfertile group ( range from 50 to 59 ) ( table 3 ). however , the test for marker - trait association ( table 8 ) still shows a significant link between the snps and the fertility trait ( p = 0 . 0124 ). once again , the haplotype ( a - a - c - a - a - t - a - a - c - t ) is strongly associated to subfertile animals ( table 10 ) ( p & lt ; 0 . 0001 ). the comparison between low nrr and subfertile also identified two haplotypes that are associated to the low nrr ( p & lt ; 0 . 0001 ) ( table 9 ). binary haplotype association analysis — high nrr vs low . although there are no significant allelic association and marker - trait association between the high and low nrr groups ( table 10 ), we still identified two haplotypes that are significantly associated to the high fertility ( p & lt ; 0 . 0001 ), and two haplotypes that are significantly associated to the low fertility ( p & lt ; 0 . 0001 ) ( table 11 ). binary haplotype association analysis — high nrr vs infertile the ld test for allelic association between the high nrr group and infertile group , once again , indicated that there is a very significant ld among the 10 snps and their alleles ( p & lt ; 0 . 0001 ) ( table 12 ). because only one infertile animal was used in the association study , the statistical tests for marker - trait and haplotype - trait associations could not be completed . however , this is not inconsistent with the fact that the haplotype is associated with the fertility trait as the infertile animal posses some unique haplotypes . “ oneway anova ” analysis of association between genotypes and the fertility trait . based on information in table 5 , we have carried out a “ one - way anova ” analysis to study the relationship between the genotypes and the quantitative measurement of nrr . fig1 shows the oneway anova analysis results with a significant statistical support ( p = 0 . 0252 ), indicating that the bdazl gene is strongly correlated to the fertility trait . “ oneway anova ” analysis of association between parental haplotype 1 and the fertility trait . similarly , we have carried out a “ oneway anova ” analysis to study the relationship between the parental haplotypes and the quantitative measurement of nrr ( fig2 and 3 ). we found that the parental haplotype 1 is significantly correlated to the nrr measurements ( fig2 ), but not the parental haplotype 2 ( fig3 ). summary of the haplotype - trait association study . the ld test for allelic association indicated that there is a very significant ld among the 10 snps and their alleles . the marker - trait association test indicated that the snps from the bdazl gene are significantly associated to the fertility trait . the test for haplotype - trait association indicated that different haplotypes are very significantly associated to the fertility trait . we found that one haplotype ( a - a - c - a - a - t - a - a - c - t ) ( seq id no . 3 ) is unique to the subfertile animals , and several haplotypes either linked to the high , low nrr , subfertile , or infertile . the bdazl snps and their haplotypes can be used in mas for sire early selection for the male fertility trait . diagnostic kits for male fertility selection using the bdazl snps . once snps from the bdazl gene have been , identified , validated and trait - association analyzed , a set of the most important snps , as described herein , that have a major impact on the genetic variation of the fertility trait can be selected . pcr primers to genotype the snps will be collected together . methods for selecting nucleic acids useful as probes and primers for detection of snps and haplotypes are known in the art . for example , u . s . patent applications 2005 / 0123929 and 2006 / 0211006 describe such methods . these patent applications are incorporated by reference herein in their entirety to describe such methods . extension of the dazl gene research to other major farm animals . the fact that orthologous genes ( boule , xdazl , daz and dazl ) in fly , frog , mouse and human have a similar function , and the success of the human daz and dazl transgenes to partially rescue the mouse dazl null phenotype indicate that the structures and functions of the daz / dazl proteins are highly conserved among species . this fact raised the possibility that dazl may also play a fundamental role in spermatogenesis in bovine and other mammalian species as well . the research findings described herein not only confirm that the bovine dazl gene does play an essential role in male fertility in bovine , indicating a similar role in other mammals , but also have demonstrated that snps and combinations of snps in the dazl gene are associated with decreased or enhanced fertility in animals normally considered to be fertile . these results indicate that analogous snps in the dazl genes in pig , sheep , horse , goats and other farm animals as well as in other domesticated animals that are subjected to breeding programs , such as dogs , exhibit similar association and can be used for identifying males with enhanced fertility . based on the results in hand , dazl gene sequences and mrna in various animals , particularly mammals , e . g ., pig , sheep , horse , goat , and dog , will be conserved in structure and highly conserved in sequence to the human , mouse and bovine dazl . while the specific snps in a given gene or genome region are not typically conserved among species , snps in the dazl regions identified herein , particularly in the introns and regulatory sequences of the gene of other animals , will exhibit similar function to those identified in bdazl herein for the identification of males with enhanced fertility as discussed herein . based on the information provided herein and the availability of dazl gene sequences of a given animal species , snps exhibiting the function of the snps herein can now be readily identified and used as described herein to identify haplotypes and genotypes associated with enhanced male fertility . such snps , haplotypes and genotypes can be used as described herein to identify males of various non - human animals , particularly those of non - human mammals , and more particularly those of horses , pigs , sheep , goats and dogs . the reproductive process is central to beef productivity . one of the most important components in the animal reproductive process is male fertility . however , male ( such as bull ) fertility has not been studied at the molecular genetic level . our study aimed to address the male infertility / subfertility problem in beef cattle with emphasis on a candidate gene bdazl . the results have provided significant implications and indications for identifying enhanced male fertility in bovine and other non - human animals . functional studies of the daz / dazl gene in fly , frog , mouse and human all support a hypothesis that the bdazl gene plays an important role in male spermatogenesis and fertility in cattle and other farm animal species . results from this study have tested this hypothesis and provided direct evidence in the function of this gene in bull spermatogenesis and fertility . the results of this study extend , however , to demonstrating that sequence variations in this gene are associated with quantitatively measurable differences in fertility of males otherwise considered to be fertile . the identification of snps from bdazl and the confirmation of the snp haplotype - fertility trait association have lead to the design of marker assisted selection ( mas ) to select sires at an early age in breeding . this , in turn , will significantly reduce the breeding cost , as males with subfertility or infertility are usually not identified until the age when they are expected to breed . in human , snp haplotype - diseases association study is becoming more common . the diagnostic purpose is to genetically screen the patient to prevent or diagnose the diseases early . in short , it is a case - by - case study in humans . in animal breeding , the main goal is to improve the entire population or the breeding value of a population by selecting animals with a favorable genotype and eliminating animals with an unfavorable genotype . the top 10 and bottom 10 bulls from semex alliance are not only normal bulls , but the best bulls in canada ( perhaps in the world ). the haplotypes we found in this study not only allow us to identify animals that are infertile or subfertile , but also allow us to select animals that have a high nrr or low nrr . the latter is even more important for ranchers and animal breeders to select animals with the best performance . the designation and application of a diagnostic kit for male fertility selection is not limited to cattle . additional studies in other farm animal species as described above , based on information provided herein , will yield further species - specific diagnostic kits . a portion of the work described herein has been published as liu , w .- s ., wang , a ., uno , y ., galtz , d ., beattie , c . w ., ponce de león , f . a . ( 2007 ) genomic structure and transcript variants of the bovine dazl gene . cytogenetic and genome research 116 ( 1 - 2 ), 65 - 71 ( see the examples below ). this reference is specifically incorporated by reference herein in its entirety to provide additional details of the cloning and analysis of bdazl . it is recognized by those skilled in the art that dna sequences may vary due to the degeneracy of the genetic code and codon usage . additionally , it will be recognized by those skilled in the art that allelic variations may generally occur in the dna sequences which will not significantly change activity of the amino acid sequences of the peptides which the dna sequences encode or affect phenotype of the organism . ( noting however that this invention relates to certain specific snps associated with significant changes in phenotype ). all such equivalent dna sequences are included within the scope of this invention . it will be understood ti the context of this invention and the nucleic acid molecules that for applications herein that the sequence of certain regions of the nucleic acid molecules of this invention is associated with the function and use of the nucleic acid molecules . variations in sequence in such regions which are functional in applications herein are not preferred in this invention and in general not result in functionally equivalent nucleic acid molecules . hybridization procedures are useful for identifying polynucleotides with sufficient homology to the subject sequences to be useful as taught herein . the particular hybridization techniques is not essential to the subject invention . as improvements are made in hybridization techniques , they can be readily applied by one of ordinary skill in the art . some of the nucleic acid molecules of this invention are useful as probes and as illustrated specifically herein as primers for pct techniques . a probe and sample are combined in a hybridization buffer solution and held at an appropriate temperature until annealing occurs . thereafter , the membrane is washed free of extraneous materials , leaving the sample and bound probe molecules typically detected and quantified by autoradiography and / or liquid scintillation counting . as is well known in the art , if the probe molecule and nucleic acid sample hybridize by forming a strong non - covalent bond between the two molecules , it can be reasonably assumed that the probe and sample are essentially identical , or completely complementary if the annealing and washing steps are carried out under conditions of high stringency . the probes detectable label provides a means for determining whether hybridization has occurred . alternatively , hybridization may be detected by virtue of priming in a polymerase chain assay . one of ordinary skill in the art understands in general how to employ nucleic acid molecules as probes pr primers without resort to undo experimentation . various degrees of stringency of hybridization can be employed . the more stringent the conditions , the greater the complementarity that is required for duplex formation . stringency can be controlled by temperature , probe concentration , probe length , ionic strength , time , and the like . preferably , hybridization is conducted under moderate to high stringency conditions by techniques well know in the art , as described , for example in keller , g . h ., m . m . manak ( 1987 ) dna probes , stockton press , new york , n . y ., pp . 169 - 170 , hereby incorporated by reference . polymerase chain reaction ( pcr ) is a repetitive , enzymatic , primed synthesis of a nucleic acid sequence . this procedure is well known and commonly used by those skilled in this art [ see , e . g ., mullis , u . s . pat . nos . 4 , 683 , 195 , 4 , 683 , 202 , and 4 , 800 , 159 ; saiki et al . ( 1985 ) science 230 : 1350 - 1354 ]. pcr is based on the enzymatic amplification of a dna fragment of interest that is flanked by two oligonucleotide primers that hybridize to opposite strands of the target sequence . the primers are oriented with the 3 ′ ends pointing towards each other . repeated cycles of heat denaturation of the template , annealing of the primers to their complementary sequences , and extension of the annealed primers with a dna polymerase result in the amplification of the segment defined by the 5 ′ ends of the pcr primers . since the extension product of each primer can serve as a template for the other primer , each cycle essentially doubles the amount of dna template produced in the previous cycle . this results in the exponential accumulation of the specific target fragment , up to several million - fold in a few hours . by using a thermostable dna polymerase such as the taq polymerase , which is isolated from the thermophilic bacterium thermus aquaticus , the amplification process can be completely automated . other enzymes which can be used are known to those skilled in the art . as used herein percent sequence identity of two nucleic acids is determined using the algorithm of altschul et al . ( 1997 ) nucl . acids res . 25 : 3389 - 3402 ; see also karlin and altschul ( 1990 ) proc . natl . acad . sci . usa 87 : 2264 - 2268 , modified as in karlin and altschul ( 1993 ) proc . natl . acad . sci . usa 90 : 5873 - 5877 . such an algorithm is incorporated into the nblast and xblast programs of altschul et al . ( 1990 ) j . mol . biol . 215 : 402 - 410 . blast nucleotide searches are performed with the nblast program , score = 100 , wordlength = 12 , to obtain nucleotide sequences with the desired percent sequence identity . to obtain gapped alignments for comparison purposes , gapped blast is used as described in altschul et al . ( 1997 ) nucl . acids . res . 25 : 3389 - 3402 . when utilizing blast and gapped blast programs , the default parameters of the respective programs ( nblast and xblast ) are used . see the national center for biotechnology information on the internet . when a markush group or other grouping is used herein , all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure . a number of specific groups are described herein . it is intended that all combinations and subcombinations of the specific groups of defined are individually included in this disclosure . all known variants of nucleic acids are encompassed herein . the invention includes isotopic variants , including radioisotopes of molecules described herein . molecules described herein can be radiolabelled or otherwise labeled or tagged as known in the art for use in assays herein . methods for making such isotopic , radiolabelled and tagged variants are known in the art . for example , tagging technology such as taqman ® systems ( applied biosystems ), and sybr ® green ( invitrogen ) are frequently used in snp analysis . every formulation or combination of components described or exemplified herein can be used to practice the invention , unless otherwise stated . whenever a range is given in the specification , for example , a temperature range , a time range , or a composition or concentration range , all intermediate ranges and subranges , as well as all individual values included in the ranges given are intended to be included in the disclosure . it will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the claims herein . all patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains . references cited herein are incorporated by reference herein in their entirety to indicate the state of the art as of their publication or filing date and it is intended that this information can be employed herein , if needed , to exclude specific embodiments that are in the prior art . for example , when composition of matter are claimed , it should be understood that compounds known and available in the art prior to applicant &# 39 ; s invention , including compounds for which an enabling disclosure is provided in the references cited herein , are not intended to be included in the composition of matter claims herein . as used herein , “ comprising ” is synonymous with “ including ,” “ containing ,” or “ characterized by ,” and is inclusive or open - ended and does not exclude additional , unrecited elements or method steps . as used herein , “ consisting of ” excludes any element , step , or ingredient not specified in the claim element . as used herein , “ consisting essentially of ” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim . when the broad term “ comprising ” is used herein , it is intended to encompass the narrower terms “ consisting essentially of ” and “ consisting of .” thus , the phrase “ comprising a and b ” encompasses the narrower phrase “ consisting essentially of a and b ” and the even narrower phrase “ consisting of a and b .” the use of the term comprising herein is intended to also provide support for “ consisting essentially of ” and “ consisting of .” the invention illustratively described herein suitably may be practiced in the absence of any element or elements , limitation or limitations which is not specifically disclosed herein . one of ordinary skill in the art will appreciate that starting materials , biological materials , reagents , synthetic methods , purification methods , analytical methods , assay methods , and biological methods other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation . all art - known functional equivalents , of any such materials and methods are intended to be included in this invention . the terms and expressions which have been employed are used as terms of description and not of limitation , and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof , but it is recognized that various modifications are possible within the scope of the invention claimed . thus , it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features , modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art , and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims . all references cited herein are hereby incorporated by reference to the extent that there is no inconsistency with the disclosure of this specification . some references provided herein are incorporated by reference to provide details concerning sources of starting materials , additional starting materials , additional reagents , additional methods of synthesis , additional methods of analysis , additional biological materials , additional nucleic acids , chemically modified nucleic acids , additional cells , and additional uses of the invention . the deleted in azoospermia - like gene ( dazl ) is a member of the deleted in azoospermia ( daz ) family , which consists of three genes : boll , daz and dazl . it is believed that the ancestor member of the family is boll , which gave rise to dazl via duplication prior to the divergence of vertebrates and invertebrates ( cauffman et al ., 2005 ). later , during primate evolution , the autosomal dazl gene gave rise to daz on the y chromosome by transposition , repeat amplification and pruning ( saxena et al ., 1996 ; shan et al ., 1996 ; gromoll et al ., 1999 ). as a result , daz has been identified only in old world monkeys and great apes , while dazl is present in all vertebrates ( cooke et al ., 1996 ; saxena et al ., 1996 ). the daz family is expressed exclusively in the germ cells encoding proteins that contain a highly conserved rna - recognition motif ( rrm ) and a unique daz repeat of 24 amino acid ( aa ) residues ( reviewed by yen , 2004 ). daz genes are expressed only in the testis ( reijo et al ., 1995 ), while dazl is expressed in both the testis and the ovary ( seligman and page , 1998 ; dorfman et al ., 1999 ; cauffman et al ., 2005 ). both daz and dazl are detected in the nuclei of primordial germ cells ( pgcs ) in fetal gonads ( xu et al ., 2001 ), and are believed to function in the development of pgcs and in germ cell differentiation and maturation ( reviewed by yen , 2004 ). mutations in these genes have been linked to infertility in several species . in flies , loss of function of the boule gene , an ortholog of daz / dazl , leads to male sterility ( eberhart et al ., 1996 ). in frogs , inhibition of xdazl ( xenopus daz - like ) leads to defective migration and a reduction in pgcs ( houston and king , 2000 ). in mice , a disruption of dazl leads to prenatal loss of all germ cells in both sexes during prenatal germ cell development , and hence infertility ( ruggiu et al ., 1997 ). further , the infertile phenotype of dazl null mouse ( dazl —/—) can be partially rescued by a human daz transgene ( slee et al ., 1999 ). as the gene name suggests , deletion or microdeletion in daz gene ( s ) on the y chromosome leads to severe spermatogenic failure and infertility in men ( reijo et al ., 1995 ; ferlin et al ., 1999 ), and dazl transcripts in the testes are lower in men with spermatogenic failure compared with fertile men ( lin et al ., 2001 ). a polymorphism ( a / g transition ) occurring in the rna binding domain of dazl confers susceptibility to severe spermatogenic failure in humans , providing direct evidence for the role of the dazl gene in human spermatogenesis ( teng et al ., 2002 ). however , the dazl gene has not been characterized in any farm animal species . to examine the role of the dazl gene in fertility / infertility and to facilitate the isolation of potential single - nucleotide polymorphisms ( snps ) from dazl for bull fertility selection , we have cloned and characterized the bovine dazl ( bdazl ) gene . we have found that the bdazl is highly conserved with the human and mouse sequence , maps to the distal region of the bovine chromosome ( bta ) 1 , and is expressed only in the testis in cattle . materials and methods . development of a daz gene probe and isolation of bovine bac clones containing dazl . we designed a pair of pcr primers from human daz2 mrna ( genbank acc . no . nm — 020363 ) ( table 13 ), that amplified a 120 bp fragment from genomic dna of man , to generate a probe for screening the testis cdna library and the bovine rpci - 42 bac library ( bac / pac resources center , calif .) for the bovine dazl . the pcr products were labeled with isotope 32p , and used as a probe to screen the male bovine bac library . the pcr products amplified from the positive bac clones were sequenced at the advanced genetic analysis center ( agac ), university of minnesota ( umn ), to confirm the presence of gene sequences in the positive clones . bovine testis cdna library construction and screening total rna was extracted from an adult testis of a healthy bull ( chomczynski and sacchi , 1987 ), and mrna was isolated with the poly a tract isolation system ( promega , madison , wis .) according to the manufacturer &# 39 ; s protocol . in order to obtain a library with larger inserts and higher titer , three cdna libraries ( s / l1 , s / l3 , and e / l3 ) were constructed with different ratios of vector - to - cdna insert during ligation , using the zap express cdna synthesis kit ( stratagene , la . jolla , calif .). the e / l3 library was screened with the human daz2 probe . positive clones were isolated and the phagemid dna was purified by a miniprep kit ( perkin elmer , foster city , calif .) and sequenced at agac , umn . the bac dnas from clones 304i5 , 94b4 , 162l5 and 248k19 that were positive for the daz2 probe were labeled with biotin - 14 - datp using the gibco brl bionick ™ labeling kit . metaphase chromosome slides were prepared using standard procedures from peripheral blood lymphocytes obtained from a normal , healthy bull , and fish experiments were carried out essentially as described ( liu and ponce de leon , 2004 ). the genomic structure of bdazl was analyzed using a pcr strategy in combination with a blast search approach against the bovine genome sequence ( build 2 . 1 ) ( http :// www . ncbi . nlm . nih . gov / genome / seq / btablast . html ). primers flanking the possible introns were designed from the dazl cdna sequence using primer3 ( http :// frodo . wi . mit . edu / cqi - bin / primer3 / primer3_www . cgi ) ( rozen and skaletsky , 2000 ) ( table 13 ). pcr with different combinations of primer pairs was performed in a 96 - well techne touchgene thermocycler using the dazl positive bac clone or genomic dna as templates with the dazl cdna clone and the testis cdna libraries as positive controls . each pcr reaction contained 20 ng of dna , 1 × buffer ( takara , biomedicals ), 2 mm mgcl2 , 200 μm dntps , 5 μmol of each primer and 0 . 5 u takara ex taq dna polymerase in a total volume of 20 μl . the amplification cycle included denaturation at 95 ° c . for 5 min , followed by 36 cycles at 94 ° c . for 1 min , 52 - 64 ° c . for 1 min , 72 ° c . for 1 - 7 min depending upon the intron size , and a final extension step at 72 ° c . for 7 min . pcr products containing any intron sequences were sequenced at agac , umn . total rna was extracted from 13 different bovine tissues , including testis , ovary , lung , liver , kidney , spleen , cerebellum , adrenal , longissimus , lymphnode , thymus , semitendon and peripheral blood mononuclear cells ( pbmc ), using trizol reagent ( gibco / life technologies , grand island , n . y .) as per the manufacturer &# 39 ; s instruction . the purity and integrity of the rna samples were verified as described ( pelle and murphy , 1993 ). for rt - pcr analysis , 1 to 5 μg of total rna in 10 μl of rnase - free water was incubated at 70 ° c . for 10 min in the presence of 1 μg of random hexamers or oligo ( dt ) 15 ( invitrogen , carlsbad , calif .) and 2 . 5 mm of each dntp . the reaction sample was chilled immediately and the following components were added for a final volume of 20 μl : 5 × first strand buffer , 0 . 1 mm dithiothreitol ( dtt ), 40 u of rnase inhibitor and 200 u of superscript iiirnase h — reverse transcriptase ( invitrogen , carlsbad , calif .). the reaction was incubated at 50 ° c . for 50 min and finally heated at 85 ° c . for 5 min to deactivate the transcriptase activity . two μl aliquots from the first - strand cdna synthesis reaction were then amplified in a 50 μl reaction containing 1 × pcr buffer , 1 . 5 mm mgcl2 , 200 μm dntps , 5 μmol of each primer ( dazl416f and dazl621r ) and 2 . 5 u of taq polymerase . the amplification cycle included an initial 95 ° c ., 7 min denaturing step , followed by 35 cycles consisting of 94 ° c . for 1 min , 60 ° c . for 1 min , and 72 ° c . for 1 min , and a final extension step at 72 ° c . for 7 min . the pcr products were analyzed by electrophoresis in a 1 . 5 % agarose gel stained with ethidium bromide . d dazl primer series were designed from the bovine dazl cdna isolated in this study . the numeral numbers after dazl stand for the position in the cdna . e primers designed from the mouse gapdh mrna , and amplified a 116 bp fragment . b as the 5 ′ utr in the cdna is not present in the region of 88 , 265 - 69 , 473 in the genomic contig , it means the start position of the intron 1 is excluded from the contig , indicating that the intron 1 is larger that 18 , 793 bp . cloning and sequence analysis of bdazl . we screened the bovine testis cdna library e / l3 with the human daz2 pcr probe and isolated four positive cdna clones . one of the positive clones , namely 11 . 1a . 3 , was selected for further analysis . sequencing of this cdna clone indicated that it contains a full - length mrna that is 1 , 373 bp long ( genbank acc . no . dq408765 , fig4 ). blast search against the ncbi database http :// www . ncbi . nlm . nih . gov / blast /) showed that it is the bovine homolog of the human daz / dazl and mouse dazl gene . the cdna includes a 146 bp 5 ′ untranslated region ( utr ), an open reading frame ( orf ) for a protein of 295 aa residues with an rna recognition motif ( rrm ), and a 339 bp 3 ′ utr fig4 ). compared to the human daz / dazl sequences , the bdazl coding region is closer to human dazl ( 90 % identical ) than the daz cluster ( 84 - 89 %). the latter contains 7 - 15 copies of a tandem repeat of 24 aa ( 72 bp ) ( so - called daz repeat ) in its coding region . like human dazl , bdazl contains only one daz repeat at position of nt 645 - 716 ( fig4 ). the bdazl cdna also has a high similarity to the mouse dazl ( 91 %). the sequence alignment further revealed that there is a breakpoint at nt 1 , 083 in the 3 ′ utr of bdazl , where a 1 , 577 bp sequence ( nt 1 , 228 - 2 , 805 of nm — 001351 ) is present in the human dazl 3 ′ utr . a similar size fragment of 1 , 589 bp ( nt 1 , 119 - 2 , 708 of nm — 010021 ) is also present in the mouse dazl 3 ′ utr . the deduced protein sequence of bdazl contains 295 aa , identical in length to the human dazl protein ( np — 001342 ), but three aa shorter than that of the mouse ( np — 034151 ). the peptide sequence of bdazl is 97 % similar to mouse dazl , while 96 % similar to human dazl . the rrm region is located at aa 32 - 117 ( fig4 ), which is completely conserved between bovine and mouse . one amino acid substitution ( y to f ) at the position of aa 88 is found in the rrm when compared to human dazl ( fig4 ). there are several entries in the genbank for bdazl , including a gene locus ( loc530116 ), an mrna ( xm — 608581 ) and a protein ( xp — 608581 . 2 ), all predicted from the recently released bovine genome sequence ( build 2 . 1 ). when we compared the full length cdna and the deduced aa sequence obtained in this study , we found that the predicted mrna ( xm — 608581 ) is a partial mrna , and the predicted protein ( xp — 608581 . 2 ) is different from the actual protein sequences of human , mouse and bovine dazl in both the n - and c - terminals . alternative rna splicing is found in the bdazl 3 ′ utr . to address questions as to why the mrna of the bdazl gene is much shorter than that of the human and mouse , and whether alternative splicing is responsible for the absence of the ˜ 1 . 6 kb sequence in the bdazl 3 ′ utr , we performed pcrs with dna templates from the bovine genomic dna , the bovine testis cdna libraries and the dazl cdna clone using primer pair dazl1055f / 1237r ( table 13 ). this primer pair , flanking the breakpoint ( nt 1 , 083 ), is predicted to amplify a 183 bp product from all dna templates examined . when pcr products were separated on a 1 % agarose gel ( not shown ), the primer pair was shown to amplify the predicted product from the cdna clone . however , the primer amplified an ˜ 1 . 8 kb fragment instead of a 183 bp fragment from genomic dna ( both bac dna and male bovine genomic dna ). the primer pair amplified both fragments from the three testis cdna libraries , indicating that there are two types of transcripts for bdazl , which differ from each other in the 3 ′ utr . rt - pcr with the same primer pair on the testis rna sample isolated from another bull confirmed the presence of the two types of transcripts . the ratio of the 1 . 8 kb to the 183 bp products varied among the three testis cdna libraries . the e / l3 library had the highest amount of transcripts for the small fragment , so did the rt - pcr products , which suggests that the shorter transcript predominated . sequencing the pcr products indicated that the small fragment corresponded to the cdna sequence , while the large fragment contained an additional 1 , 623 bp insertion in the position nt 1 , 083 of the cdna ( fig4 ). the inserted sequences are 88 and 84 % identical to the corresponding region in the human and mouse dazl 3 ′ utr , respectively . here we refer to the longer transcript ( 2 , 996 bp ) as bdazl transcript variant 1 ( genbank acc . no . dq408764 ), and the shorter transcript ( 1 , 373 bp ) as bdazl transcript variant 2 ( genbank acc . no . dq408765 ). as the dazl1055f / 1237r primer pair amplified only the 1 . 8 kb fragment corresponding to variant 1 from bovine genomic dna , we presume that only one copy of the bdazl gene is active in the bovine genome . taken together , these observations strongly suggest that the two transcripts are probably the products of alternative rna splicing of the bdazl gene . a report that two dazl transcripts , ˜ 3 . 5 kb and ˜ 2 . 3 kb , are expressed at different levels in the testis of cynomologus monkey ( carani et al ., 1997 ) suggested that the ˜ 3 . 5 kb is the predominant transcript . it is not clear from the report which region the two transcripts share and which region is missing from the short version . transcript variants are also found in the testis for the human daz cluster . for example , daz2 has three variants ( ferlin et al ., 2004 ), which vary in the daz repeats of the coding region . the finding of the transcript variants in the 3 ′ utr of bdazl in the present study is important from a gene regulatory point of view . many mrnas undergo translational regulation , normally mediated by sequences within the utrs . previous studies on the zebra fish dazl ( zdazl ) gene identified a sequence motif ‘ guuc ’ in the zdazl 3 ′ utr that can activate the translation of its own mrna ( maegawa et al ., 2002 ). we compared the sequences of 3 ′ utr from both variants to determine whether such a motif is present , and if there is a difference in copy number between the two bdazl transcript variants . we found one ‘ guuc ’ motif ( starting at nt 2 , 899 in fig4 ) common to both transcripts and four additional ‘ guuc ’ motifs ( nt 1 , 228 ; 2 , 247 ; 2 , 537 ; and 2 , 626 ; fig4 ) within the 1 , 623 bp fragment unique to the transcript variant 1 . according to the results of maegawa et al . ( 2002 ), the level of dazl translational activation is directly proportional to the copy number of ‘ guuc ’ motifs . as such , bdazl transcript variant 1 may be more active than the transcript variant 2 in protein translation . genomic organization of the bdazl gene . initially , a pcr strategy was used to analyze the genomic structure of the bdazl gene . we used different combinations of pcr primers ( table 13 ) to amplify the gene fragments using dazl positive bac clones , male bovine genomic dna , the dazl cdna and bovine testis cdna libraries ( as control ). we sequenced all pcr products that amplified from the genomic dna and were considered to contain intron sequence . we were able to identify the positions and sizes of exons 1 to 5 and introns 2 to 5 ( table 14 , fig5 ). by sequencing the bac 30415 using primer dazl118f and dazl231r , we determined that intron 1 is inserted after the start codon at nt 149 of the bovine cdna . as intron 1 is large , routine and long - pcr with primer pair dazl118f / 231r failed to determine the intron size . to completely determine the genomic structure of the bdazl gene , we then blast searched the bovine genome sequence ( build 2 . 1 ) ( http :// www . ncbi . nim . nih . gov / genome / seq / btablast . html ) using the bdazl cdna . the search yielded two sequences , one of which was a bos taurus chromosome un genomic contig ( btun_wga7606 ), with an e - value of 3e - 136 . multiple sequence alignment among the btun_wga7606 , the dazl cdna and the sequence contig for the exons 1 - 5 and introns 2 - 5 generated by pcr in this study indicated that this genomic contig contains the bdazl gene except for the 5 ′ utr and exon 1 region . it confirmed our pcr mapping results for the exons 2 - 5 and intron 2 - 5 at a 100 % identity and further revealed the existence of additional exons ( 6 - 11 ) and introns ( 6 - 10 ) in the bdazl gene ( table 14 , fig5 a ). it also confirmed that the additional 1 , 623 bp in the transcript variant 1 , spliced out from the transcript variant 2 at nt 1 , 083 of the bdazl cdna , is present in the genomic contig ( nt 54 , 951 - 56 , 573 bp ) ( fig4 , fig5 a ). the results demonstrated that the bdazl gene structure is very similar to human dazl and is composed of 11 exons and 10 introns ( suppl . table 2 , suppl . fig2 ). exon size ranges from 51 to 147 bp , while the size of introns 2 - 10 ranges from 92 to 4 , 859 bp ( table 14 ). even with this genomic contig , the precise size of intron 1 still could not be determined . since the 5 ′ utr plus the start codon ( nt 1 - 149 ) in the bovine cdna do not match any sequence in the nt 69 , 473 - 88 , 265 region in the genomic contig btun_wga7606 , we estimated that the intron 1 in bdazl is larger than 18 , 793 bp . therefore , the bdazl gene spans at least 33 kb in the genome ( fig5 a ). by comparison , both human ( fig5 b ) and mouse dazl have 11 exons and 10 introns , and span ˜ 19 and 14 kb , respectively . furthermore , as the genomic contig btun_wga7606 does not contain the entire bdazl gene ( fig5 a and b ), the incorrect prediction of the bdazl mrna and protein in the bovine genome sequence project is anticipated . bdazl maps to bta1q . in order to map bdazl , we screened the male bovine bac library ( rpci - 42 ) with the dazl probe and isolated four positive bac clones . bac finger printing analysis indicated that the four positive bacs overlapped forming a contig ( data not shown ). all four bac clones were labeled with biotin dutp and fish mapped on bovine male metaphase chromosome spreads . strong and distinct hybridization signals were observed at the distal region of the long arm of bta1 for all four bac probes ( data not shown ). no signal was observed on other chromosomes including the x and y ( fig6 ). in the human , besides the four copies of daz on the y chromosome , the autosomal gene dazl maps on hsa3 ( 3p24 . 3 ) at nt 16 , 622 , 010 - 16 , 603 , 307 ( build 35 , fig6 ). the recently published cattle - human whole genome comparative maps ( everts - van der wind et al ., 2005 ; itoh et al ., 2005 ) demonstrated that bta1 is conserved with segments of hsa21 and hsa3 , including the hsa3 sequence of nt 15 , 457 , 380 - 18 , 770 , 186 homologous to the distal region of bta1q ( fig6 ), in agreement with the fish results , mapping bdazl to bta1q . mapping this gene to an autosome in cattle further confirms previous work that all mammals except for humans , great apes and old world monkeys , do not have a daz gene on their y - chromosomes , but instead have a dazl gene on an autosome ( saxena et al ., 1996 ; skaletsky et al ., 2003 ). in bovine genome sequence build 2 . 1 , the unmapped sequence contig btun_wga7606 was not associated with any bac clone . mapping of the bdazl gene to this genomic contig in the present study provides direct evidence for placing this contig at the distal region of bta1q in future assemblies of the bovine genome sequence . furthermore , four bac clones were isolated for the dazl gene from the bovine rpci - 42 bac library . bac fingerprint analysis indicated that these bacs form a contig that spans about 300 kb . comparing the bac contig with the genomic contig btun_wga7606 in future studies will allow identification of any overlapped regions . sequencing the non - overlapped region ( s ) from the bac contig will serve two purposes . first , determine the size of intron 1 for the dazl gene ; second , close the gap for bta1 in that region . even if the bac contig is not large enough to close the gap , it will provide a starting point for closure of that gap in future sequence assembly . a dazl pseudogene is present on bta16 . during the analysis of the bdazl genomic structure , primer pair dazl602f / 1074r amplified a 473 bp fragment ( fig7 ) across all dna samples examined . when this fragment was sequenced , we found that the sequence amplified from the genomic dna was only 87 % identical to the corresponding sequence amplified from the cdna clone and cdna libraries , raising the question whether there are two copies of the dazl gene in the bovine genome . as mentioned above , the blast search against the bovine genome sequence ( build 2 . 1 ) with the bdazl cdna resulted in two hits : one was the genomic contig ( btun_wga7606 ), described above , that contains the dazl gene , the other was a bos taurus chromosome 16 genomic contig ( bt16_wga2706 ). surprisingly , the latter had a lower e - value of 0 . 0 with a score of 652 , suggesting that this genomic contig most likely contains a copy of the bdazl gene . further sequence comparison showed that the bta16 genomic contig does contain a segment that is homologous to the entire bdazl cdna ( fig7 ). the overall sequence identity between the two sequences is ˜ 87 %. while a 13 bp ( nt 384 - 397 in the bdazl cdna ) fragment is deleted from the nw — 929036 genomic sequence at position nt 157 , 758 , two large insertions are found in the genomic sequence ( fig7 ). the first insertion is 874 bp ( nt 158 , 035 - 158 , 909 ) long , referring to a position between nt 247 and 279 in the cdna . the second insertion is 2 , 522 bp ( nt 154 , 555 - 157 , 077 ), which is inserted exactly at the position of the break point ( nt 1 , 083 ) in the cdna ( fig7 ). although the 2 , 522 bp fragment is 899 bp larger than the 1 , 623 bp fragment obtained from the dazl transcript variant 1 , the two sequences show 78 - 91 % identity from region to region , although several gaps exist . the deletion and the first insertion occur in locations or responding to the rrm region in dazl ( fig4 ), probably leading to a loss of function . these results indicated that the dazl homologous sequence in bta16 is almost certainly a duplicate of the dazl cdna , originated through a reverse transcription mechanism . bovine dazl gene is expressed in testis only . expression of bdazl mrna was analyzed by rt - pcr in 13 different tissues including both male and female gonads . primer pair dazl416f / 621r ( table 13 ) flanking exon 4 and exon 6 was expected to amplify a 206 bp fragment from the rna samples and a 743 bp from genomic dna . a housekeeping gene glyceraldehyde 3 - phosphate dehydrogenase ( gapdh ) was used as an internal control . ti was observed that while the gapdh is expressed across all tissues analyzed , the expected 206 bp rtpcr product was amplified only from the testis rna and the three testis cdna libraries , indicating that the bdazl gene is expressed in testis only . the spatiotemporal expression of the dazl gene varies among species investigated . human dazl , like the daz cluster , was found to be testis - specific ( yen et al ., 1996 ), but later was found expressed in the ovary ( seligman and page , 1998 ; dorfman et al ., 1999 ; cauffman et al ., 2005 ) and in pgcs in fetal gonads ( xu et al ., 2001 ). mouse dazl is also expressed in both male and female gonads ( cooke et al ., 1996 ). in the cynomologus monkey , dazl was considered to be expressed only in the testis ( carani et al ., 1997 ). however , transcripts from the ovary were not included in the northern blot analysis ( carani et al ., 1997 ). in the present study , rt - pcr revealed that bdazl is expressed only in the testis . in conclusion , we report the cloning of the bdazl cdna and the deduced aa sequences , which revise the prediction from the bovine genome sequence ( build 2 . 1 ). we further demonstrated that dazl is expressed only in the bovine testis and maps to the distal region on bta1q . additional bdazl is highly conserved with the human and mouse dazl in mrna , protein and even in the genomic organization levels . agulnik a i , zharkikh a , boettger - tong h , bourgeron t , mcelreavey k and bishop c e ( 1998 ) evolution of the daz gene family suggests that y - linked daz plays little , or a limited , role in spermatogenesis but underlines a recent african origin for human populations . hum mol genet 7 ( 9 ): 1371 - 1377 . burgoyne p s ( 1996 ) fruit ( less ) flies provide a clue . nature 381 ( 6585 ): 740 741 . cauffman g , van de velde h , liebaers i and van steirteghem a ( 2005 ) dazl expression in human oocytes , preimplantation embryos and embryonic stem cells . mol hum reprod 11 ( 6 ): 405 - 411 . cooke h j , lee m , kerr s and ruggiu m ( 1996 ) a murine homologue of the human daz gene is autosomal and expressed only in male and female gonads . hum mol genet . 5 ( 4 ): 513 - 516 . eberhart c g , maines j z and wasserman s a ( 1996 ) meiotic cell cycle requirement for a fly homologue of human deleted in azoospermia . nature 381 ( 6585 ): 783 - 785 . ferlin a , moro e , garolla a and foresta c ( 1999 ) human male infertility and y chromosome deletions : role of the azf - candidate genes daz , rbm and dffry . hum reprod 14 ( 7 ): 1710 - 1716 . gromoll j , weinbauer g f , skaletsky h , schlatt s , rocchietti - march m , page d c and nieschlag e ( 1999 ) the old world monkey daz ( deleted in azoospermia ) gene yields insights into the evolution of the daz gene cluster on the human y chromosome . hum mol genet . 8 ( 11 ): 2017 - 2024 . houston d w and king m l ( 2000 ) a critical role for xdazl , a germ plasm - localized rna , in the differentiation of primordial germ cells in xenopus . development 127 ( 3 ): 447 - 456 . lin y m , chen c w , sun h s , tsai s j , hsu c c , teng y n , lin j s and kuo p l ( 2001 ) expression patterns and transcript concentrations of the autosomal dazl gene in testes of azoospermic men . mol hum reprod 7 ( 11 ): 1015 - 1022 . maegawa s , yamashita m , yasuda k and inoue k ( 2002 ) zebrafish daz - like protein controls translation via the sequence ‘ guuc ’. genes cells 7 ( 9 ): 971 - 984 . reijo r , lee t y , salo p , alagappan r , brown l g , rosenberg m , rozen s , jaffe t , straus d , hovatta o and et al . 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