Characterization of baboon pregnancy-specific beta 1-glycoprotein

Immunostaining of baboon placental tissues with anti-human pregnancy-specific beta 1-glycoprotein (SPI) antibodies demonstrated that an SP1-like molecule was present in the syncytiotrophoblasts. Staining was observed on the membrane and in the cytoplasm, but the nucleus was devoid of any staining. Western blot analysis further demonstrated the presence of five protein species in baboon placental extract, whereas four protein bands were detected in human placental extract. Culture medium of baboon placental villi also contained five SP1-like molecules with sizes slightly different from those present in the placental extract. Amniotic fluid and culture medium of decidua basalis and chorioamniotic tissue contained lesser quantities and fewer species of SP1-like molecules. However, an 87 kDa band was present in all samples examined. Northern blot analysis of baboon placenta with a human placental SP1 cDNA probe demonstrated the presence of a 1.65 Kb band, whereas two hybridizing bands (1.65 Kb and 2.25 Kb) were present in human placenta. Southern blot analysis of baboon genomic DNA further demonstrated the presence of multiple bands hybridizing with a human placental SP1 cDNA probe. These results showed the presence in baboons of multiple genes encoding mRNAs and proteins highly similar to human placental SP1.     

DNA fragments from regions involved in surface antigen expression specifically identify Listeria monocytogenes serovar 4 and a subset thereof: cluster IIB (serotypes 4b, 4d, and 4e).

Listeria monocytogenes serotype 4b has frequently been implicated in sporadic as well as epidemic listeriosis. On the basis of pulsed-field fingerprinting, serotype 4b strains, along with strains of serotypes 4d and 4e, constitute one genomic cluster (IIB). We have identified two genomic regions essential for the expression of surface antigens which previously were shown to be specific to cluster IIB strains. A DNA probe of 1.1 kb derived from one of the regions (probe 1) hybridized only with strains of serotypes 4b, 4d, and 4e in Southern blots and dot blots. A different DNA probe of 0.3 kb (probe 2), derived from the other region, hybridized with all serovar 4 strains (serotypes 4b, 4a, 4c, 4d, and 4e). All other L. monocytogenes serotypes were negative with probe 1 or 2. Use of probe 1 in Southern blots of EcoRI-digested genomic DNA revealed a restriction fragment length polymorphism in serotype 4b strains, with the hybridizing EcoRI fragments being 4.5 kb (strains of the epidemic clone) and either 4.5 or 5.0 kb (all other serotype 4b strains). Although the probes hybridized with a special group of Listeria innocua strains which also expressed the surface antigens, the latter could be readily distinguished by the size of the hybridizing EcoRI fragment with probe 1 (ca. 2.2 kb). These data suggest that the combined use of these probes with L. monocytogenes can readily and specifically identify cluster IIB strains as well as the entire serovar 4 complex.     

(TG)n uncovers a sex-specific hybridization pattern in cattle

Screening of a bovine genomic library with the human minisatellite 33.6 probe uncovered a family of clones that, when used to probe Southern blots of bovine genomic DNA digested with the restriction enzyme HaeIII or MboI, revealed sexually dimorphic, but otherwise virtually monomorphic, patterns among the larger DNA fragments to which they hybridized. Characterization of one of these clones revealed that it contains different minisatellite sequences. The sexual dimorphism hybridization pattern observed with this clone was found to be due to multiple copies of two tandemly interspersed repeats: the simple sequence (TG)n and a previously undescribed 29-bp sequence. Both repeats appear to share many genomic loci including autosomal loci. In contrast, Southern analysis of AluI- or HinfI-digested bovine DNA with the (TG)n repeat used as a probe yielded substantial polymorphism. These results show that (i) different minisatellites can be found in a cluster, (ii) both simple and more complex repeated sequences other than the simple quaternary (GATA)n repeat can be sexually dimorphic, and (iii) simple repeats can reveal substantial polymorphism.     

A polymerase chain reaction-based method for isolation of gene-specific sequences from the interferon-alpha gene cluster.

The interferon-alpha gene is a gene family of over 20 distinct genes having 80-95% homology with one another at a nucleotide level. Because of the high homology in the gene cluster, the available interferon-alpha gene probes can hybridize to multiple bands of different size on Southern blot analysis of restricted human genomic DNA. We used the polymerase chain reaction with the primers synthesized from Alu repetitive sequence and the conserved sequences of the interferon-alpha gene cluster to generate specific probes for individual interferon-alpha genes. The amplification products were subcloned into a plasmid vector and analyzed by DNA sequencing and Southern blotting of the restricted human placental DNA. One clone, which derived from interferon-alpha 14 gene, produced a single 5.2-kb band in Southern blots of the HindIII-restricted human placental DNA. This stands in contrast to the 10 bands of different size that were detected with a cDNA for the interferon-alpha I' gene. Our results indicate that a polymerase chain reaction-based method can be used to isolate gene-specific sequences from the interferon-alpha gene cluster. Since a variety of human cancers has been found to have the complete or partial deletion of the interferon-alpha gene cluster, the gene-specific probe generated by this method may aid in determining the breakpoints in the vicinity of the gene cluster.     

Variation in the Major Urinary Protein Multigene Family in Wild-Derived Mice

The levels of expression and genomic organization of genes coding for the major urinary proteins (MUPs) were examined in several stocks of wild-derived mice. Levels of MUP mRNA in the liver varied considerably with M. musculus Brno and M. castaneus males having several-fold more MUP RNA than inbred C57BL/6 males, whereas M. hortulanus, M. caroli and M. cervicolor displayed levels much lower than C57BL/6. Analysis of RNA with MUP cDNAs specific to two different subfamilies of MUP genes revealed that M. caroli and M. cervicolor primarily expressed a MUP mRNA that was less abundant in C57BL/6, suggesting differential expression of subfamilies of genes within the MUP multigene complex. Although inbred males usually have five-fold more MUP mRNA than inbred females, male to female ratios for wild-derived stocks ranged from one to several hundred. Southern blots of genomic DNA hybridized to MUP subfamily probes revealed differences in restriction fragment sizes as well as possible variation in the number of MUP genes in some species. Analysis of urinary proteins from hybrids between C57BL/6 and M. spretus suggested that low MUP expression in M. spretus females was due to cis-acting genetic elements.     

Identification of a Ferritin Light Chain Pseudogene Near the Glycerol Kinase Locus in Xp21 by cDNA Amplification for Identification of Genomic Expressed Sequences

We used cDNA amplification for identification of genomic expressed sequences (CAIGES) to identify genes in the glycerol kinase region of the human X chromosome. During these investigations we identified the sequence for a ferritin light chain (FTL) pseudogene in this portion of Xp21. A human liver cDNA library was amplified by vector primers, labeled, and hybridized to Southern blots ofEcoRIdigested human genomic DNA from cosmids isolated from yeast artificial chromosomes in the glycerol kinase region of Xp21. A 3.1-kb restriction fragment hybridized with the cDNA library, was subcloned and sequenced, and a 440-bp intronless sequence was found with strong similarity to the FTL coding sequence. Therefore, the FTL pseudogene that had been mapped previously to Xp22.3–21.2 was localized specifically to the glycerol kinase region. The CAIGES method permits rapid screening of genomic material and will identify genomic sequences with similarities to genes expressed in the cDNA library used to probe the cloned genomic DNA, including pseudogenes.     

Characterization of cDNAs of the human pregnancy-specific beta 1-glycoprotein family, a new subfamily of the immunoglobulin gene superfamily

Three highly homologous cDNAs encoding human pregnancy-specific beta 1-glycoprotein (SP1) were isolated from a human placental cDNA library. These cDNAs share greater than 90% nucleotide homology in their coding sequences, and greater than 79% of the encoded amino acids are homologous. Proteins encoded by these cDNAs are very similar to members of the carcinoembryonic antigen family and contain repeating domains, conserved disulfide bridges, and beta-sheet structure typical of the immunoglobulin gene superfamily. However, the high degree of sequence homology and relatively lesser degree of glycosylation among the SP1 proteins suggest that they exist as a unique family instead of being members of the CEA family. Both soluble and potentially membrane-bound forms of SP1 proteins were present in the placenta. Northern blot analysis using specific probes confirmed the expression of multiple mRNA species in human term placenta.     

Polymorphic class II sequences linked to the rat major histocompatibility complex (RT1) homologous to human DR and DQ sequences

Until recently, the analysis of Class II genes linked to the rat major histocompatibility complex, RT1, has been confined to serologic and electrophoretic analysis of their gene products. To obtain a more definitive estimate of the number and relative polymorphism of RT1 Class II sequences, we performed Southern blot analysis of rat genomic DNA employing human cDNA probes specific for Class II heavy and light chain genes. Southern blots of EcoRI and BamHI digests of genomic DNA from ten inbred strains, expressing eight RT1 haplotypes, were hybridized with the human DQ beta or DR beta cDNA that are homologous to Class II light chain sequences. Four to eight bands were observed to hybridize with the light chain cDNA: band sizes ranged from 2.5 to 28 kb. Restriction fragment patterns were polymorphic; the only identical patterns observed were those associated with RT1 haplotypes with identical RT1.B regions. The number and size of bands hybridizing with DQ beta and DR beta suggested a minimum of four light chain sequences in each haplotype. Southern blots of BamHI and EcoRI digests of genomic DNA from the same strains were hybridized with a DR alpha cDNA that is homologous to Class II heavy chain sequences. All RT1 haplotypes expressed either a 10.0-kb or 13.0-kb band when digested with BamHI, and either a 17-kb or 3.7-kb band when digested with EcoRI. Considerably less polymorphism was detected with the DR alpha probe; this observation is consistent with previously reported limited protein polymorphism of the rat equivalent of the I-E alpha subunit. The size and number of bands hybridizing with the DR alpha probe suggests a minimum of two heavy chain sequences. These observations suggest that the RT1 complex includes more Class II sequences than have been observed in serologic and electrophoretic analyses of Class II gene products. Furthermore, the level of polymorphism of RT1 Class II sequences appears to be comparable with mouse and human Class II sequences.     

Somatic cell mapping of bovine EC-SOD and SOD1L loci.

cDNA probes of human extracellular superoxide dismutase (EC-SOD) and bovine superoxide dismutase 1 (SOD1) genes were hybridized to Southern blots containing genomic DNAs from cow-rodent somatic cell lines segregating bovine chromosomes. The SOD1 probe identified two loci: the coding locus (SOD1), which mapped to bovine U10; and a related locus (SOD1L), which mapped to U11. EC-SOD mapped to bovine U15. The mapping of EC-SOD to human chromosome 4, and our mapping of EC-SOD to U15, further defines a region of extensive syntenic conservation between humans and domestic cows.     

Conservation of human Y chromosome sequences among male great apes: Implications for the evolution of Y chromosomes

Nine newly described single-copy and lowcopy-number genomic DNA sequences isolated from a flow-sorted human Y chromosome library were mapped to regions of the human Y chromosome and were hybridized to Southern blots of male and female great ape genomic DNAs (Gorilla gorilla, Pan troglodytes, Pongo pygmaeus). Eight of the nine sequences mapped to the euchromatic Y long arm (Yq) in humans, and the ninth mapped to the short arm or pericentromeric region. All nine of the newly identified sequences and two additional human Yq sequences hybridized to restriction fragments in male but not female genomic DNA from the great apes, indicating Y chromosome localization. Seven of these 11 human Yq sequences hybridized to similarly-sized restriction endonuclease fragments in all the great ape species analyzed. The five human sequences that mapped to the most distal subregion of Yq (deletion of which region is associated with spermatogenic failure in humans) were hybridized to Southern blots generated by pulsed-field gel electrophoresis. These sequences define a region of approximately 1 Mb on human Yq in which HpaII tiny fragment (HTF) islands appear to be absent. The conservation of these human Yq sequences on great ape Y chromosomes indicates a greater stability in this region of the Y than has been previously described for most anonymous human Y chromosomal sequences. The stability of these sequences on great ape Y chromosomes seems remarkable given that this region of the Y does not undergo meiotic recombination and the sequences do not appear to encode genes for which positive selection might occur. Correspondence to: B. Steele Allen     

Detection of DNA sequence polymorphisms in human genomic DNA by using denaturing gradient gel blots.

Denaturing gradient gel electrophoresis can detect sequence differences outside restriction-enzyme recognition sites. DNA sequence polymorphisms can be detected as restriction-fragment melting polymorphisms (RFMPs) in genomic DNA by using blots made from denaturing gradient gels. In contrast to the use of Southern blots to find sequence differences, denaturing gradient gel blots can detect differences almost anywhere, not just at 4-6-bp restriction-enzyme recognition sites. Human genomic DNA was digested with one of several randomly selected 4-bp recognition-site restriction enzymes, electrophoresed in denaturing gradient gels, and transferred to nylon membranes. The blots were hybridized with radioactive probes prepared from the factor VIII, type II collagen, insulin receptor, beta 2-adrenergic receptor, and 21-hydroxylase genes; in unrelated individuals, several RFMPs were found in fragments from every locus tested. No restriction map or sequence information was used to detect RFMPs. RFMPs can be used as genetic markers, because their alleles segregate in a Mendelian manner. Unlike most other methods for detecting DNA sequence polymorphisms, a genomic DNA blot made from one gel can be hybridized consecutively with many (30 or more) different probes.     

Human ovary and placenta express messenger RNA for multiple activin receptors

In the present study, we examined the expression of activin receptor (ActR) mRNAs in human ovary and placenta. Primers specific for two type I and two type II activin receptors (ActR-I, ActR-IB, ActR-II, and ActR-IIB) were used in polymerase chain reaction (PCR) to amplify cDNAs prepared from granulosa-luteal cells, placental tissues and isolated trophoblast cells. PCR products with the expected sizes for ActR-I, ActR-IB, ActR-II, and ActR-IIB mRNAs were detected in freshly dissociated and 5-day cultured granulosa-luteal cells; and in trophoblast cells from both first trimester and term placentas. The identity of these PCR products were confirmed by Southern blot hybridization, as well as cloning and sequencing. These results suggest that multiple activin receptors are present in human ovary and placenta and may mediate activin function in these tissues. The demonstration of activin receptor mRNAs in granulosa-luteal and trophoblast cells further supports the notion that activin is an important local regulator in the human ovary and placenta.     

The human sex hormone-binding globulin gene contains exons for androgen-binding protein and two other testicular messenger RNAs

When a sex hormone-binding globulin (SHBG) cDNA was used to screen a human testicular cDNA library, three distinct cDNAs were isolated, one of which corresponds to the human SHBG cDNA sequence and probably represents testicular androgen-binding protein. The other two SHBG-related cDNAs each contain unique 5' regions that diverge from the SHBG cDNA sequence at the same position, and one of them (SHBGr-2) lacks a 208-base pair region within the SHBG cDNA. As a result, this cDNA could potentially encode for a truncated form of SHBG which lacks N-linked carbohydrates and part of the steroid-binding domain. Southern blots of human placental DNA and cloned genomic DNA fragments also indicate that SHBG and its related testicular cDNAs are the products of a single gene. Sequence analysis of the gene indicates that the complete coding region for the SHBG precursor is comprised of 8 exons, which are distributed over 3.2 kilobase (kb) of genomic DNA, and the unique 5' regions associated with the two SHBG-related testicular cDNAs were identified 1.9 kb upstream from the initiating codon for SHBG. In addition, the deletion within SHBGr-2 is due to the removal of exon 7, and an interesting feature of the gene is that differentially used exons are preceded by Alu repetitive DNA sequences. Although the relative abundance of the various SHBG-related mRNAs in the testis has not been established, Northern blot analysis indicates that they are similar in size (1.6 kb) to that of hepatic SHBG mRNA.