New type of POU domain in germ line-specific protein Oct-4.

Members of a family of murine octamer-binding proteins interact specifically with the octamer motif, a transcription regulatory element found in the promoter and enhancer regions of many genes. Oct-4 is a maternally expressed protein that is also present in the pre-implantation mouse embryo. Although many regulatory proteins are expressed in post-implantation embryos, transcription factors regulating pre-implantation processes have remained elusive. The Oct-4 gene is therefore a prime candidate for an early developmental control gene. Here we report the complementary DNA cloning of the mouse Oct-4 gene, and the characterization of the encoded protein(s) by sequential in vitro transcription, translation, DNA-binding and protease-clipping analysis. Deletion analysis shows that the DNA-binding activity is mediated by a POU domain encoded in an open reading frame corresponding to a 324-amino-acid protein. Sequence comparison with known POU domains reveals that Oct-4 is a novel member of the POU-family.

A family of octamer-specific proteins present during mouse embryogenesis: evidence for germline-specific expression of an Oct factor.

We have analysed various adult organs and different developmental stages of mouse embryos for the presence of octamer-binding proteins. A variety of new octamer-binding proteins were identified in addition to the previously described Oct1 and Oct2. Oct1 is ubiquitously present in murine tissues, in agreement with cell culture data. Although Oct2 has been described as a B-cell-specific protein, similar complexes were also found with extracts from brain, kidney, embryo and sperm. In embryo and brain at least two other proteins, Oct3 and Oct7, are present. A new microextraction procedure allowed the detection of two maternally expressed octamer-binding proteins, Oct4 and Oct5. Both proteins are present in unfertilized oocytes and embryonic stem cells, the latter containing an additional protein, Oct6. Whereas Oct4 was not found in sperm or testis, it is expressed in male and female primordial germ cells. Therefore Oct4 expression is specific for the female germline at later stages of germ cell development. Our results indicate that a family of octamer-binding proteins is present during mouse development and is differentially expressed during early embryogenesis. Protease clipping experiments of Oct4 and Oct1 suggest that both proteins contain similar DNA-binding domains.

Heterogeneity in the human Ig VH locus.

We have used synthetic oligonucleotides corresponding to human VH sequences to analyze sequence variation in human genomic DNA. By using probes 20 to 24 bp long and conditions of hybridization and washing under which identity in 17 to 21 consecutive bp is required for hybridization, it has been possible to dramatically reduce the complexity of hybridization patterns. We have been able to identify unambiguously individual VH elements. Concomitant with the reduction in overall complexity of hybridization patterns has been a marked increase in the variation between hybridization patterns when different individuals are compared. Variation between individuals was detected using probes corresponding to both framework and complementarity determining regions and depended in part on the complexity of the corresponding VH gene family. Probes corresponding to a cDNA clone belonging to the single-member VH6 family, hybridized to a single, invariant, band in all individuals tested. An oligonucleotide probe corresponding to CDR2 of one member of the VH3 family also detected a single, invariant, band in all individuals tested. However, an oligonucleotide probe corresponding to framework region 2 revealed variants of more than 40% of the 22 VH elements it detects. In addition, a panel of 5 oligonucleotide probes corresponding to a second member of the VH3 family revealed variants of 10 of 14 elements detected. The patterns of variation suggest that some VH elements have multiple alleles, whereas some elements are remarkably conserved. The number of variant elements we have detected is evidence that the haplotype arrangement of the human VH locus is probably extremely complex. Importantly, this heterogeneity may contribute directly to disease susceptibility in man.

Studies of human germinal mutations by deoxyribonucleic acid hybridization.

Spontaneous mutations that occur in human germ cells contribute significantly to clinical disorders and result in premature mortality, incurable morbidity, mental handicap, and infertility. We have used molecular analysis of deoxyribonucleic acid to study the occurrence of spontaneous human germinal mutations. We examined 458 offspring and parents in 60 multigeneration human families. Probes for hypervariable loci were dispersed throughout all chromosomes to note the occurrence of new mutations. We found that both point mutations and insertion-deletion mutations occur frequently enough to be directly quantitated. The rates of occurrence detected at the molecular level are much greater than the rates detected with other modalities. The mutational rates at some loci approach 1% in live-born children. Such mutations appear to be sequence specific and related to the processes of meiosis or mitosis as they occur in the production of human gametes.

Seasonal variations in testicular germ cell stages and in plasma concentrations of sex steroids in male rainbow trout (Salmo gairdneri) maturing at 2 years old.

Male rainbow trout of an autumn- and a spring-spawning strain were sampled every 28 days over a period of 2 years, starting when they were approximately 6 months old. Plasma concentrations of testosterone (T), 11-ketotestosterone (11-K), and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) were measured by radioimmunoassay. A histometric method was used to determine the absolute quantities of several germ cell stages, including spermatogonia B, spermatocytes, spermatids, spermatozoa, and resorbing spermatozoa. The general pattern of gonadal development in the two strains was similar, e.g., steroids peaking in the order T----11-K----17,20 beta-P. However, there were a large number of differences--in the timing of first appearance of various parameters, in the timing and magnitude of various peaks, in the histological appearance of the regressing testes, and in the heterogeneity of the male population. T and 11-K levels were not associated with any particular germ cell stage in the two strains. Peak 17,20 beta-P levels coincided with the period when ovulated females were present and milt production was maximal.

Differentiation and development of the reproductive system in the iguanid lizard, Sceloporus undulatus.

Embryos of the lizard Sceloporus undulatus were sampled throughout incubation, and the differentiation and development of the reproductive system was documented histologically. The undifferentiated gonads possess both a cortex and medulla, both of which contain germ cells until embryonic stage 34. Beginning at stage 34, the cortex of the presumptive ovary thickens, and cortical germ cells are more abundant. By the time of hatching, the ovarian cortex is 6 to 10 cells thick and filled with oogonia and oocytes; primordial follicles, however, are not yet present. In males at embryonic stage 34, seminiferous tubules appear in the medulla of the testis, and Sertoli cells begin to differentiate. Seminiferous tubule formation is complete by hatching, and both Sertoli and Leydig cells are apparent. The mullerian ducts develop in both sexes but begin regressing in the male at embryonic stage 37. The wolffian ducts also develop in both sexes and are present in males and females at hatching.

Genetic engineering of bovine Ig. Construction and characterization of hapten-binding bovine/murine chimeric IgE, IgA, IgG1, IgG2, and IgG3 molecules.

A bovine recombinant phage library was constructed and screened with rabbit S mu and human C gamma probes. IgM, IgA, and IgE H chain constant region (CH) genes were isolated with the rabbit S mu probe and three IgG CH genes were isolated with the C gamma probe. The CH genes were individually cloned into an expression vector which contained a murine VDJ gene cloned from a hybridoma producing anti-dansyl hapten antibody. The resulting constructs were transfected into murine hybridoma cells producing L chain of the anti-dansyl antibody and stable transfectomas secreting chimeric bovine-murine IgA, IgE, or IgG subclass anti-dansyl antibodies were obtained. The chimeric antibodies, immunoprecipitated with Ag or with anti-bovine H chain antibodies, were analyzed by SDS-PAGE and were shown to contain H and L chains of expected size. Of the three chimeric antibodies derived from the C gamma genes, one reacted with anti-IgG1 antibody, another reacted with anti-IgG2 antibody and the third did not react with either anti-IgG1 or anti-IgG2. This third IgG appears to represent a "new" subclass of bovine IgG, IgG3. Southern blot analysis indicated that the bovine genome contains a fourth C gamma gene. These experiments demonstrate the usefulness of molecular genetic techniques for the isolation and characterization of Ig which are not readily purified from biologic fluids. These techniques will be useful for isolation and characterization of Ig genes from other outbred mammals.

Partial molecular genetic map of the rabbit VH chromosomal region.

Thirty VH-containing cosmid clones, isolated from rabbit germ-line DNA libraries, were restriction mapped and shown to contain approximately 100 VH genes in 765-kb of DNA. Twenty-two of the cosmid clones were grouped into seven distinct clusters. The VH genes were separated by an average of 8 kb, although some were separated by less than 3 kb. Comparison of the nucleotide sequences of two of these VH genes with the sequences of another 11 VH genes showed that they were all generally more than 80% homologous suggesting that rabbit VH genes are members of one highly homologous gene family. Most rabbit Ig molecules have the VH allotypic specificities a1, a2, or a3 and are designated VHa-positive. A small number (less than 30%) of Ig molecules lack these VHa allotypic specificities and are designated VHa-negative. The VH containing cosmid clones were hybridized with synthetic oligomer probes designed to be specific for genes encoding VHa-positive or VHa-negative molecules. At least 50% of the germ-line VH genes hybridized with the VHa-negative oligomer and thus presumably encode VHa-negative molecules; as few as 15% of the genes could be identified as encoding VHa-positive molecules based on hybridization with the VHa-positive oligomer. Approximately 35% of the VH genes did not hybridize with either oligomer and could not be classified as VHa-negative or VHa-positive. We propose that the predominance of serum VHa-positive molecules, in contrast to the predominance of VHa-negative encoding germ-line genes, may reflect preferential usage of a few germline VH genes. The implications of this idea toward explaining the allelic inheritance of VHa allotypes are discussed.

I.29 lymphoma cells express a nonmutated VH gene before and after H chain switch.

The I.29 B cell lymphoma consists of IgM+ and IgA+ cells which express the same germ-line VH gene. IgA+ cells of the I.29 lymphoma were derived from the IgM+ cells by a typical H chain switch recombination event. The IgM+ cells can be induced with LPS to undergo H chain switching in culture. It has been proposed that the somatic hypermutation process is activated during H chain switch, since V genes expressed in IgG+ and IgA+ cells have more frequently undergone mutation than those expressed in IgM+ cells. We have investigated this question by sequencing VH genes expressed before and after H chain switch in the I.29 lymphoma. We have also sequenced the germ-line VH gene corresponding to the gene expressed by I.29 cells to determine whether the VH gene expressed in the IgM+ cells had already undergone somatic mutation. Our results indicate that somatic mutation was not activated in the precursor cell for the I.29 lymphoma, nor during isotype switch in I.29 cells. It is possible that cells of the I.29 lymphoma, or their precursor, have not received the signal which induces somatic mutation, or that I.29 cells belong to a subset of B cells that cannot be induced to undergo any (or much) somatic mutation.

Detection and differentiation of glycoconjugates in various cell types by lectin histochemistry.

Knowledge of chemical structure of glycoconjugates (GCs) at precise loci has increased through histochemical use of a battery of horseradish peroxidase-conjugated lectins, each possessing affinity for a specific terminal sugar or internal sugar linkage. Lectin histochemistry has shown tremendous variability among GCs in different histologic sites, reflecting known chemical diversity of these substances. GCs differ in structure among various cell types in an animal and differ at a given histologic site between species or between individuals in outbred but not inbred species. Lectin conjugates react with and detect GCs not otherwise demonstrable histochemically and, because of low concentration in tissue, not identified biochemically. Lectin-HRP conjugates have visualized unique GC with terminal GalNAc in primordial germ cells of rat embryos, with terminal Gal in epithelial basal cells of rodents and nodes of Ranvier in rats and with terminal GalNAc in a cell population in the thymus, Peyer's patches and intestinal lamina propria of some but not other mice.

Expression of the caudal gene in the germ line of Drosophila: formation of an RNA and protein gradient during early embryogenesis.

The caudal (cad) gene of Drosophila encodes a maternal and a zygotic transcript which have different promoters. Both mRNAs contain the same open reading frame, including a homeo box. In situ hybridization and antibody staining show that the maternal RNA and protein are localized in an anteroposterior gradient during the syncytial blastoderm stage. The protein is found mainly in the nuclei and is also present in the pole cells. Zygotic RNA and protein are localized in the primordia of the terminal abdominal segment, the hindgut, and in the posterior midgut rudiment. In third instar larvae, cad is expressed in the gut, the gonads, and parts of the genital discs. It is the first homeo box-containing gene expressed in the germ line of Drosophila.

Migratory and postmigratory mouse primordial germ cells behave differently in culture.

In all vertebrate groups, the progenitors of the germ line, the primordial germ cells (PGCs) arise extragonadally and move to the developing gonad early in embryonic development. We have examined the behavior of isolated pregonadal and gonadal PGCs in vitro on feeder layers of an embryo-derived cell line. Histochemically and serologically identified pregonadal germ cells are found to be actively motile in vitro and, furthermore, show behavior characteristic of invasive cells. PGCs isolated from the developing gonad, however, show little locomotory activity and are not invasive on the same cellular substrate. These observations suggest that PGCs undergo a major change in phenotype at the time of their entry into the gonad anlagen.

Stage specific embryonic carbohydrate surface antigens of primordial germ cells in mouse embryos: FAL (S.S.E.A.-1) and globoside (S.S.E.A.-3).

Immunodetections of carbohydrate surface antigens were carried out for SSEA-1 and SSEA-3. Using alkaline phosphatase for the detection of primordial germ cells these surface antigens were detected at the cell membrane and the cytoplasm of the germ cells at E 10.

Characterization of the purified Chlamydomonas minus agglutinin.

Chlamydomonas flagellar sexual agglutinins are responsible for the adhesion of opposite mating-type (plus and minus) gametes during the first stages of mating. Purification and partial characterization of the plus agglutinin was previously reported (Adair, W. S., C. J. Hwang, and U. W. Goodenough, 1983, Cell, 33:183-193). Here we characterize the purified minus molecule. We show it to be a high molecular weight, hydroxyproline-rich glycoprotein that migrates in the 3% stacking region of an SDS-polyacrylamide gel and is absent from two nonagglutinating minus mutants. Plus and minus agglutinins are remarkably similar, although nonidentical, in amino acid composition, molecular morphology, and reactivity in vivo and in vitro with monoclonal antibodies raised against the plus agglutinin. Moreover, the adhesiveness of both plus and minus agglutinins, when coupled to agarose beads, is abolished by thermolysin, trypsin, periodate, alkaline borohydride, reducing agents, or heat, but unaffected by exo- or endoglycosidases. The minus agglutinin, however, migrates just ahead of the plus molecule on SDS PAGE, is excluded from an anion-exchange (Mono Q) column, elutes earlier during hydrophobic interaction (Bio-gel TSK Phenyl 5PW) chromatography, and is sensitive to chymotrypsin digestion (unlike the plus agglutinin); therefore, it differs from the plus agglutinin in apparent molecular weight, net charge, relative hydrophobicity and proteolytic susceptibility. Nevertheless, our results generally demonstrate a high degree of homology between these complementary cell-cell recognition/adhesion molecules, which suggests that they are specified by genes that have a common evolutionary origin.

Studies of foreign genes transmitted through the germ lines of transgenic mice.

A novel gene transfer system has recently been developed in which DNA sequences isolated by molecular cloning can be introduced and permanently integrated into the genomes of developing mice. The procedure entails the microinjection of such cloned sequences into the pronuclei of one-celled embryos followed by reimplantation into pseudopregnant females for continued development. Although data obtained from this system are still few, they indicate it will have broad applicability to problems of mammalian developmental genetics. In order that such experiments be fully exploited, it will be necessary to understand the behavior of foreign genes introduced into the mouse and the relationship of that behavior to endogenous gene structure, function, and regulation. As a step in this direction, we have examined a line of mice into which has been transferred a recombinant plasmid consisting of a human leukocyte interferon genomic fragment cloned into the BamHI site of pBR322. The donor material has been integrated as a large concatameric structure which is inherited as a single Mendelian trait to several generations of progeny. Crossing of heterozygotes has apparently resulted in the production of viable homozygotes. In one animal, the structure of the integrated fragment was altered in spleen DNA but not in germ cells. This finding may reflect a normal processing mechanism whereby the structure of genes is altered during cell differentiation, or it may mean that transferred DNA does not behave as endogenous genes do.

A comparative study of the lipid composition of isolated rat Sertoli and germinal cells.

The lipid composition of enriched preparations of Sertoli cells and of germinal cells, isolated from the testes of mature rats, has been investigated. Sertoli cells contained a much lower content of phospholipids (in particular, much less phosphatidylcholine and phosphatidylethanolamine) and a higher content of triacylglycerols than did germinal cells. In addition, the Sertoli cells had a higher ratio of esterified to unesterified cholesterol than did germinal cells. Total lipids of Sertoli cells contained considerably lower levels of palmitic and docosa-4,7,10,13,16-pentaenoic acids and higher levels of stearic and oleic acid than did the total lipids of germinal cells. The major phospholipid classes and the triacylglycerols of Sertoli cells similarly contained less palmitic and docosa-4,7,10,13,16-pentaenoic acids, more stearic and oleic acids and also more arachidonic acid than did the corresponding lipid classes of the germinal cells. Minor differences between cell types were also noted for the content of palmitoleic, linoleic, docosa-7,10,13,16-tetraenoic, docosa-4,7,10,13,16,19-hexaenoic and tetracosa-9,12,15,18-tetraenoic acids.

[Investigation on fat-quality of embryo, endosperm and crush fractions of maize kernel (author's transl)]. / Untersuchungen über die Fettqualität von Keimling, Endosperm und technologischen Mahlfraktionen des Maiskornes

Investigations were conducted on the composition of fatty acids in the fat of the embryo, the endosperm and several crush fractions of the maize kernel. The following results were obtained: 1. The embryo fat contained relatively lower contents of palmitin-, stearin- and linolenic acid, on the other hand higher contents of oleic- and linoleic acid in comparism to endosperm fat. 2. This result was identical to each one of the seven varieties examined. 3. The examined varieties presented clear differences both in the absolute contents of single fatty acids in the embryo and in the endosperm, as well as in the relationship between the components in the germ and in the endosperm. 4. The differences observed between the germ- and the endosperm fat in the crush fractions in some cases were clearly visible, while in other cases were not traceable.