Christa Bode

Histones: genetic diversity and tissue-specific gene expression.

Abstract Histones are the major protein constituents of the chromatin of eukaryotic cell nuclei. This group of basic proteins is extremely conserved throughout evolution and includes five classes termed H1, H2A, H2B, H3and H4. In mammals, each of these classes except H4 is subdivided into several subtypes. The most divergent class of histones is the H1 protein family, which consists of seven different subtypes, termed H1.1–H1.5, H1°, and H1t. The subtypes H1.2 and H1.4 are found in most somatic cell nuclei, whereas H1° is found in several differentiated tissues, and H1t is restricted to mammalian testicular cells. Similarly, core histone subtypes replacing the major forms of H2A, H2B or H3 have been described. Biochemical analysis of protein and RNA from different tissues and cell lines demonstrates varied patterns of expression of individual histone subtype genes. Moreover, antibodies against specific histone subtypes and in situ hybridization with subtype-specific probes indicate that the expression of histone subtype genes is in several cases modulated in a tissue-specific manner. This is particularly evident at the different stages of spermatogenesis when chromatin undergoes substantial reorganization, which finally results in the highly condensed state of chromatin of the mature sperm head.

Spermatogenesis proceeds normally in mice without linker histone H1t.

Abstract. The histone gene H1t is expressed exclusively in pachytene spermatocytes of the testis. In this report we have eliminated the single copy H1t gene by homologous recombination from the mouse genome to analyse the function of the H1t protein during spermatogenesis. Mice homozygous for the mutated H1t gene locus developed normally and showed no anatomic abnormalities until the adult stage. In addition, H1t-deficient mice were fertile and reproduced as wild-type mice. The process of spermatogenesis and the testicular morphology remained unchanged in the absence of H1t. RNase protection analysis demonstrated that H1.1, H1.2 and H1.4 histone gene expression is enhanced during spermatogenesis in H1t-deficient mice.

EXPRESSION OF THE MOUSE TESTICULAR HISTONE GENE H1T DURING SPERMATOGENESIS

The testicular H1 histone variant, H1t, is synthesized during spermatogenesis in mammalian male germ cells. In situ hybridization and immunohistochemical techniques were used to assign the expression of either the H1t mRNA or the H1t protein to specific cell stages of spermatogenesis. Our results show the presence of the H1t mRNA only in the late and mid-pachytene stages, whereas the protein occurs first in pachytene spermatocytes, and persists until later stages from round up to elongated spermatids.

Structure and expression of the mouse testicular H1 histone gene (H1t).

A mouse genomic library was screened with a human testicular H1 (H1t) gene fragment. One phage containing the testis specific mouse H1t histone gene and its flanking regions was isolated. Northern blot analysis showed that the mouse H1t gene is expressed only in mouse testis at the stage of pachytene spermatocytes and that the H1t mRNA is not polyadenylated. This mouse H1t gene encodes a protein which differs from the somatic mouse H1 proteins, but is similar to the known H1t proteins from rat, and man.

Isolation of two murine H1 histone genes and chromosomal mapping of the H1 gene complement

The mammalian H1 histone gene complement consists of at least seven H1 protein isoforms. These include five S-phase-dependent H1 protein subtypes and two more distantly related proteins, which are expressed upon terminal differentiation (H10) or during the pachytene stage of spermatogenesis (H1t). In the past, three replication-dependent murine H1 genes plus the H10 and H1t genes have been isolated and characterized. In this report, we describe the sequences of two more H1 genes, and we show that all five murine replication-dependent H1 genes and the H1t gene map to the region A2-3 on Chromosome (Chr) 13. This is in agreement with our previous finding that the human H1 histone gene complement maps to 6p21.3, which corresponds to the A2-3 region on the murine Chr 13. Previous reports have shown that the replication-independent H10 genes map to syntenic regions on Chrs 22 (human H10) and 15 (murine H10).

Expression of the mouse histone gene H1t begins at premeiotic stages of spermatogenesis

Abstract The gene encoding H1t, a testicular variant of histone H1, is expressed in mammals during spermatogenesis. Northern blot and in situ hybridization has detected H1t mRNA only at the stage of pachytene spermatocytes. We have extended this analysis to more sensitive approaches and demonstrate, by RNase protection and electron-microscopic in situ hybridization, that H1t mRNA is detectable even in spermatogonia. Just a faint H1t band is seen in Western blots of nuclear protein from 9-day-old mice. This indicates that the H1t gene is expressed at premeiotic stages, albeit at a low level. In contrast to H1t mRNA, the H1t protein has not been detected in spermatogonia by electron microscopy after immunogold staining.

Phosphorylation of the ribosomal protein S6 in response to secretagogues in the guinea pig exocrine pancreas, parotid and lacrimal gland

not received CAMP and @+-dependent protein phosphorylation

Human Migratory Meniscus Progenitor Cells Are Controlled via the TGF-β Pathway

Summary Degeneration of the knee joint during osteoarthritis often begins with meniscal lesions. Meniscectomy, previously performed extensively after meniscal injury, is now obsolete because of the inevitable osteoarthritis that occurs following this procedure. Clinically, meniscus self-renewal is well documented as long as the outer, vascularized meniscal ring remains intact. In contrast, regeneration of the inner, avascular meniscus does not occur. Here, we show that cartilage tissue harvested from the avascular inner human meniscus during the late stages of osteoarthritis harbors a unique progenitor cell population. These meniscus progenitor cells (MPCs) are clonogenic and multipotent and exhibit migratory activity. We also determined that MPCs are likely to be controlled by canonical transforming growth factor β (TGF-β) signaling that leads to an increase in SOX9 and a decrease in RUNX2, thereby enhancing the chondrogenic potential of MPC. Therefore, our work is relevant for the development of novel cell biological, regenerative therapies for meniscus repair.

In situ-RT-PCR and immunohistochemistry for the localisation of the mRNA of the alpha 3 chain of laminin and laminin-5 during human organogenesis

Laminin-5 is known to be an integral part of the hemidesmosome and therefore responsible for the integrity of the connection of the epithelium to the basement membrane. This is also an important mechanism during embryonic development, as documented by studies in mice. In an attempt to elucidate its implication for human development we localised the mRNA of the α3 chain of laminin with the help of in situ RT-PCR, and the laminin-5 protein immunohistochemically. We systematically investigated kidney, lung, skin and intestinal tissue of consecutive developmental stages during human embryogenesis. From gw 6.5 onwards, the mRNA of the α3 chain of laminin was found exclusively in the cytoplasm of epithelial cells of the developing kidney, lung, skin and intestine. Interestingly, in the skin and intestine from gw 8 onwards, the superficial cell layers also stained positive for the mRNA, while the protein was still only found in the dermal-epidermal and enteric basement membrane zones. In all developing organs investigated, the mRNA of the α3 chain of laminin is strictly of epithelial origin and the corresponding protein localised in the underlying basement membrane zones. Due to this discrepancy, we postulate a broader role for laminin-5 during human embryogenesis, for example, for epithelial cell development, beyond its involvement in hemidesmosome formation and cell adhesion.

Specific phosphorylation of a protein in calcium accumulating endoplasmic reticulum from rat parotid glands following stimulation by agonists involving cAMP as second messenger

Stimulation of secretion in exocrine cells by agonists involving cAMP as second messenger is associated with the phosphorylation of a specific membrane‐associated 22.4‐kDa protein (protein III) (Jahn). Here it is shown by subcellular fractionation of rat parotid gland lobules that protein III is associated with the endoplasmic reticulum. The submicrosomal fractions containing protein III, also contain the ATP‐dependent microsomal calcium pump activity. Protein III in microsomal subfractions can be phosphorylated in vitro with catalytic subunit from cAMP‐dependent protein kinase. Phosphorylated protein III contains exclusively P‐serine. Protein III can be removed from ER‐membranes with acid chloroform—methanol or Triton X‐114, but not by high salt wash indicating that it is tightly associated with the membranes. Protein III is smaller than phospholamban and, in contrast to phospholamban, resistant to heating in SDS. A relationship between phosphorylation of protein III and microsomal calcium sequestration is discussed.