Hair follicle-specific keratins and their diseases.

The human keratin family comprises 54 members, 28 type I and 26 type II. Out of the 28 type I keratins, 17 are epithelial and 11 are hair keratins. Similarly, the 26 type II members comprise 20 epithelial and 6 hair keratins. As, however, 9 out of the 37 epithelial keratins are specifically expressed in the hair follicle, the total number of hair follicle-specific keratins (26) almost equals that of those expressed in the various forms of epithelia (28). Up to now, more than half of the latter have been found to be involved in inherited diseases, with mutated type I and type II members being roughly equally causal. In contrast, out of the 26 hair follicle-specific keratins only 5 have, at present, been associated with inherited hair disorders, while one keratin merely acts as a risk factor. In addition, all hair follicle-specific keratins involved in pathologies are type II keratins. Here we provide a detailed description of the respective hair diseases which are either due to mutations in hair keratins (monilethrix, ectodermal dysplasia of hair and nail type) or hair follicle-specific epithelial keratins (two mouse models, RCO3 and Ca(Rin) as well as pseudofolliculitis barbae).

Characterization of human KAP24.1, a cuticular hair keratin-associated protein with unusual amino-acid composition and repeat structure.

In a search for genes overexpressed in human sexual hairs, several partial complementary DNA (cDNA) sequences were isolated. Screening of a human scalp cDNA library with one fragment led to the isolation of a full-length cDNA clone, which showed identity to another known sequence, termed KAP24-1 (AB09693). Bioinformatic analysis revealed that the gene for this cDNA consisted of one exon and was located ca. 86 kb away from the chromosome 21q22.1 keratin-associated protein (KAP) gene domain. RT-PCR analysis of a variety of organs showed that KAP24.1 was only present in human scalp. The KAP24.1 protein consisted of 254 amino acids, exhibited a high content of serine, proline, and tyrosine, but low cysteine content and possessed several carboxyterminal tyrosine-containing tandem decameric repeat structures. Evolutionary tree analysis showed no association to other KAP family members. In situ hybridization and indirect immunofluorescence microscopy studies using an antibody derived from KAP24.1 demonstrated specific expression in the middle/upper hair cuticle. The structure of the KRTAP24, its proximity to the chromosome 21q22.1 KAP gene domain, the presence of repeat motifs in the protein and its localization in the hair cuticle points to KAP24.1 being a novel human KAP family member.

Human hair keratin-associated proteins (KAPs).

Elucidation of the genes encoding structural proteins of the human hair follicle has advanced rapidly during the last decade, complementing nearly three previous decades of research on this subject in other species. Primary among these advances was both the characterization of human hair keratins, as well as the hair keratin associated proteins (KAPs). This review describes the currently known human KAP families, their genomic organization, and their characteristics of expression. Furthermore, this report delves into further aspects, such as polymorphic variations in human KAP genes, the role that KAP proteins might play in hereditary hair diseases, as well as their modulation in several different transgenic mouse models displaying hair abnormalities.

Characterization of new members of the human type II keratin gene family and a general evaluation of the keratin gene domain on chromosome 12q13.13.

The recent completion of a reference sequence of the human genome now allows a complete characterization of the type II keratin gene domain on chromosome 12q13.13. This, domain, approximately 780 kb in size, is present on nine bacterial artificial chromosome clones sequenced by the Human Genome Sequencing Project. The type II keratin domain contains 27 keratin genes and eight pseudogenes. Twenty-three of these genes and four pseudogenes have been previously reported. This study describes, in addition to the genomic sequencing of the K2p gene and the bioinformatic identification of four keratin pseudogenes, the characterization of cDNA corresponding to three previously undescribed keratin genes K1b, K6l, and Kb20, as well as cDNA sequences for the previously described keratin genes hHb2, hHb4, and K3. Northern analysis of the new keratins K1b, K6l, K5b, and Kb20 using mRNA of major organs as well as of specific epithelial subtypes shows singular expression of these keratins in skin, hair follicles and, for K5b and Kb20, in tongue, respectively. In addition, the obvious discrepancies between the current reference sequence of the human genome and the previously described gene/cDNA sequences for K6c, K6d, K6e, K6f, K6h are investigated, leading to the conclusion that K6c, K6d as well as K6e, K6f are probably polymorphic variants of K6a and K6h, respectively. All 26 human type II keratins found on this domain as well as K18, dtype 1 Keratin, are identified at the genomic and transcriptional level. This appears to be the total complement of functional type II keratins in humans.

The human type I keratin gene family: characterization of new hair follicle specific members and evaluation of the chromosome 17q21.2 gene domain.

In general concurrence with recent studies, bioinformatic analysis of the chromosome 17q21.2 DNA sequence found in the EBI/Genebank database shows the presence of 27 type I keratin genes and five keratin pseudogenes present on 8 contiguous Bacterial Artificial Chromosome (BAC) sequences. This constitutes the 970 kb type I keratin gene domain. Inserted into this domain is a 350 kb region harboring 32 previously characterized keratin-associated protein genes. Of the 27 keratin genes found in this region, six have not been characterized in detail. This study reports the isolation of cDNA sequences for these keratin genes, termed K25irs1-K25irs4, Ka35, and Ka36, as well as cDNA sequences for the previously reported hair keratins hHa3-I, hHa7, and hHa8. RT-PCR analysis of 14 epithelial tissues using primers for the six novel keratins, as well as for keratins 23 and 24, shows that the six novel keratins appear to be hair follicle associated. Previous expression data, coupled with evolutionary analysis studies point to K25irs1-K25irs4 probably being inner root sheath specific keratins. Ka35 and Ka36 are, based on their exon-intron structure and expression characteristics, hair keratins. In contrast, K23 and K24 appear to be epithelial keratins associated with simple/glandular or stratified, non-cornified epithelia, respectively. A literature analysis coupled with the data presented here confirms that all of the 27 keratin genes found on this domain have been characterized at the transcriptional level. Together with K18, a type I keratin gene found on the type II keratin domain, this seems to be the entire complement of functional type I keratins in humans.

Androgen regulation of the human hair follicle: the type I hair keratin hHa7 is a direct target gene in trichocytes.

Previous work had shown that most members of the complex human hair keratin family were expressed in terminal scalp hairs. An exception to this rule was the type I hair keratin hHa7, which was only detected in some but not all vellus hairs of the human scalp (Langbein et al, 1999). Here we show that hHa7 exhibits constitutive expression in medullary cells of all types of male and female sexual hairs. Medullated beard, axillary, and pubic hairs arise during puberty from small, unmedullated vellus hairs under the influence of circulating androgens. This suggested an androgen-controlled expression of the hHa7 gene. Further evidence for this assumption was provided by the demonstration of androgen receptor (AR) expression in the nuclei of medullary cells of beard hairs. Moreover, homology search for the semipalindromic androgen receptor-binding element (ARE) consensus sequence GG(A)/(T)ACAnnnTGTTCT in the proximal hHa7 promoter revealed three putative ARE motifs. Electrophoretic mobility shift assays demonstrated the specific binding of AR to all three hHa7 AREs. Their function as AR-responsive elements, either individually or in concert within the hHa7 promoter, could be further confirmed by transfection studies with or without an AR expression vector in PtK2 and prostate PC3-Arwt cells, respectively in the presence or absence of a synthetic androgen. Our study detected hHa7 as the first gene in hair follicle trichocytes whose expression appears to be directly regulated by androgens. As such, hHa7 represents a marker for androgen action on hair follicles and might be a suitable tool for investigations of androgen-dependent hair disorders.

An unusual Ala12Thr polymorphism in the 1A alpha-helical segment of the companion layer-specific keratin K6hf: evidence for a risk factor in the etiology of the common hair disorder pseudofolliculitis barbae.

Pseudofolliculitis barbae (PFB) is a common hair disorder characterized by a pustular foreign body inflammatory reaction that is induced by ingrown hairs of the facial and submental (barbea) regions after regular shaving. It occurs predominantly in black males, while it is rather rare and usually far less severe in Caucasian males. Black individuals have a higher propensity of developing PFB due to their genetic predisposition for curly hair which inherently possesses a much higher risk of growing back into the skin than straight or wavy hair. The PFB process is, however, not gender dependent nor restricted to the face, but can occur in any skin region once regular shaving, plucking, or other traumatic means of hair removal are instituted. Through a family study and a large-scale investigation of randomly sampled PFB-affected and -unaffected individuals, this study demonstrates that an unusual single-nucleotide polymorphism, which gives rise to a disruptive Ala12Thr substitution in the 1A alpha-helical segment of the companion layer-specific keratin K6hf of the hair follicle, is partially responsible for the phenotypic expression and represents an additional genetic risk factor for PFB.

Hair keratin associated proteins: characterization of a second high sulfur KAP gene domain on human chromosome 21.

Analysis of the EBI/GeneBank database using nonhuman hair keratin associated protein (KAP) gene sequences as a query resulted in the identification of two human KAP gene domains on chromosome 21, one of which, located at 21q22.1, has recently been characterized. The second domain presented here, an approximately 90 kb domain on chromosome 21q23, harbored 16 KAP genes and two KAP pseudogenes. By comparison with known sheep and mouse KAP families, these genes could be assigned to two KAP families, KAP10 and KAP12, with the KAP10 family (12 members) being distinctly larger than the KAP12 family (four members). Systematic cDNA/3' rapid amplification of cDNA ends isolation studies using human scalp mRNA led to the identification of eight KAP10 and two KAP12 cDNA sequences. In situ hybridization analyses of human anagen hair follicles using specific 3'-noncoding sequences of the various KAP10/KAP12 genes revealed mRNA expression of nearly all KAP10 and KAP12 members exclusively in a narrow region of the middle portion of the hair fiber cuticle. Bioinformatic analyses of the promoter regions of the KAP10/KAP12 genes demonstrated several enhancer elements that were present in nearly all of the KAP genes. Primary among these were binding elements for the ETS, heat shock factor, AML, and HOX families of transcription factors.

K6irs1, K6irs2, K6irs3, and K6irs4 represent the inner-root-sheath-specific type II epithelial keratins of the human hair follicle.

In this study we report on the cloning of two novel human type II keratin cDNAs, K6irs3 and K6irs4, which were specifically expressed in the inner root sheath of the hair follicle. Together with the genes of two previously described type II inner root sheath keratins, K6irs1 and K6irs2, the K6irs3 and K6irs4 genes were subclustered in the type II keratin/hair keratin gene domain on chromosome 12q13. Evolutionary tree analysis using all known type II epithelial and hair keratins revealed that the K6irs1-4 formed a branch separate from the other epithelial and hair keratins. RNA in situ hybridization and indirect immunofluorescence studies of human hair follicles, which also included the K6irs2 keratin, demonstrated that both K6irs2 and K6irs3 were specifically expressed in the inner root sheath cuticle, but showed a different onset of expression in this compartment. Whereas the K6irs3 expression began in the lowermost bulb region, that of K6irs2 was delayed up to the height of the apex of the dermal papilla. In contrast, the K6irs4 keratin was specifically expressed in the Huxley layer. Moreover, K6irs4 was ideally suited to further investigate the occurrence of Flügelzellen, i.e., Huxley cells, characterized by horizontal cell extensions that pass through the Henle layer, abut upon the companion layer, and form desmosomal connections with the surrounding cells. Previously, we detected Flügelzellen only in the region along the differentiated Henle layer. Using the Huxley-cell-specific K6irs4 antiserum, we now demonstrate this cell type to be clearly apposed to the entire Henle layer. We provide evidence that Flügelzellen penetrate the Henle layer actively and may play a role in conferring plasticity and resilience to the otherwise rigid upper Henle layer.

The in vitro assembly of hair follicle keratins: comparison of cortex and companion layer keratins.

The hair follicle consists of a complex system of multiple tissue compartments that are clearly distinguishable by their morphology and type of differentiation. We have synthesized hair follicle-specific keratins from the companion layer (K6hf, K17) and the hair cortex (Ha1, Hb3, Hb6) in Escherichia coli. The assembly of purified keratins in mixtures of K6hf/K17 and in mixtures of hair cortex keratins was compared in urea solutions, low ionic strength and physiological strength buffers, by urea melting gels, electron microscopy and analytical ultracentrifugation. Both types of keratin mixtures, keratins from the companion layer and keratins from the hair cortex, formed heterotypic complexes at 5 M urea. In low ionic strength buffers, the keratins from the companion layer were assembled to bona fide intermediate filaments. In contrast, mixtures of hair cortex keratins stayed in an oligomeric state with a mean s value of 9 as determined in sedimentation velocity experiments. Hair cortex keratins were, however, assembled into intermediate filaments at physiological salt conditions. A point mutated hair cortex keratin [Hb6(Glu402Lys)] formed no long filaments when mixed with Ha1; instead, the assembled structures showed a length distribution of 50.8 +/- 13.4 nm, comparable to the size distribution of assembly intermediates called 'unit-length' filaments.

Characterization of a first domain of human high glycine-tyrosine and high sulfur keratin-associated protein (KAP) genes on chromosome 21q22.1.

Analysis of the EBI/GeneBank(TM) data base using non-human hair keratin-associated protein (KAP) cDNA sequences as a query resulted in the identification of a first domain of high glycine-tyrosine and high sulfur KAP genes located on human chromosome 21q22.1. This domain, present on the DNA accession numbers and, was approximately 535 kb in size and contained 17 high glycine-tyrosine and 7 high sulfur KAP genes, as well as 9 KAP pseudogenes. Based on amino acid sequence comparisons of the encoded proteins, the KAP genes could be divided into seven high glycine-tyrosine gene families (KAP6-KAP8, and KAP19-KAP22) and four high sulfur gene families (KAP11, KAP13, KAP15, and KAP23). The high glycine-tyrosine genes described here appear to represent the complete set of this type of KAP genes present in the human genome. Both systematic cDNA isolation studies from an arrayed scalp cDNA library and in situ hybridization expression studies of all of the KAP genes identified in the 21q22.1 region revealed varying degrees and regions of expression of 11 members of the high tyrosine-glycine genes and 6 members of the high sulfur KAP genes in the hair forming compartment.

Polymorphisms in the human high sulfur hair keratin-associated protein 1, KAP1, gene family.

Hair fiber differentiation and maturation involves the close interaction between hair keratins and their associated proteins, KAPs. Recently, a cluster of seven human KAP multigen families has been identified on chromosome 17q12-21 among which were four hKAP1 genes (hKAP1.1B, hKAP1.3, hKAP1.4, and hKAP1.5). In addition, there were previous as well as recent reports on four additional hKAP1 genes (hKAP1.1A, hKAP1.2, hKAP1.6, and hKAP1.7) with unknown chromosomal location. In this study, we have analyzed these eight hKAP1 genes in unrelated Japanese and Caucasian individuals and discovered that hKAP1.1A, hKAP1.6, and hKAP1.7 represent size polymorphisms of the hKAP1.1B gene. In addition, we show that hKAP1.2 as well as three hitherto unknown genes (hKAP1.8A, hKAP1.8B, and hKAP1.9) are size polymorphisms of the hKAP1.3 gene. In contrast, no polymorphic alleles were found for the hKAP1.4 and hKAP1.5 genes. We provide evidence that the polymorphic hKAP1.1B and hKAP1.3 alleles arose mainly by intragenic deletion and/or duplication events of distinct pentapeptide repeats typical for hKAP1 genes. We also demonstrate the occurrence of both frequent and rare population-specific hKAP1.1B and hKAP1.3 alleles, which were obviously generated after the divergence of the Caucasian and Japanese lineage. In addition, by means of a pan-hKAP1 antibody, we confirm the previous hKAP1 family mRNA localization data in the middle to upper cortex of the human anagen hair follicle.

A novel epithelial keratin, hK6irs1, is expressed differentially in all layers of the inner root sheath, including specialized huxley cells (Flügelzellen) of the human hair follicle.

In this study we have characterized a novel human type II keratin, hK6irs1, which is specifically expressed in the inner root sheath of the hair follicle. This keratin represents the ortholog of the recently described mouse inner root sheath keratin mK6irs. The two keratins were highly related and migrated at the same height as keratin 6 in two-dimensional gel electrophoresis. Both RNA in situ hybridization and indirect immunofluorescence studies of human hair follicles demonstrated hK6irs1 expression in the Henle and Huxley layers as well as in the cuticle of the inner root sheath. In all three layers, the expression of hK6irs1 mRNA and protein began simultaneously in adjacent cells of the lowermost bulb above the germinative cell pool. Higher up in the follicle, the detection limits for both hK6irs1 mRNA and protein precisely coincided with the asynchronous onset of abrupt terminal differentiation of the Henle layer, inner root sheath cuticle, and Huxley layer. Mainly above the level of terminal Henle cell differentiation, both indirect immunofluorescence and immunoelectron microscopy revealed the occurrence of distinct Huxley cells that developed pseudopodal hK6irs1-positive extensions passing through the fully keratinized Henle layer. These outwardly protruding foot processes abutted upon cells of the companion layer, with which they were connected by numerous desmosomes. These specialized Huxley cells have previously been termed "Flügelzellen", which means "winged cells", with reference to their characteristic foot processes. We provide evidence that, together with Henle cells, Flügelzellen ensure the maintenance of a continuous desmosomal anchorage of the companion layer along the entire inner root sheath. This tightly connected companion layer/inner root sheath unit provides an optimal molding and guidance of the growing hair shaft.

Is the loose anagen hair syndrome a keratin disorder? A clinical and molecular study.

OBJECTIVES: To report the clinical features of the loose anagen hair syndrome and to test the hypothesis that the typical gap between the hair and the inner root sheath may result from hereditary defects in the inner root sheath or the apposed companion layer. DESIGN: Case series. SETTING: A pediatric dermatology unit (referral center). PATIENTS: A consecutive sample of 17 children (13 girls). For 9 of them and their first-degree relatives, molecular analyses were performed in the K6HF gene with 50 appropriate controls. INTERVENTION: Minoxidil therapy (5% lotion) in 11 patients for 1 to 12 months. MAIN OUTCOME MEASURES: Clinical and follow-up features and determination of mutations in the K6HF gene. RESULTS: Most patients had easily pluckable hair with no sign of scalp inflammation or scarring. Ten patients seldom cut their hair, and 4 had unmanageable hair. One patient had hypodontia. Two patients had an additional clinical phenotype of diffuse partial woolly hair. The family history was positive for loose anagen hair syndrome in 5 patients. Marked improvement was noted after treatment with 5% minoxidil lotion in 7 of the 11 patients treated. Polymerase chain reaction analysis of the gene segments encoding the alpha-helical 1A and 2B subdomains of K6hf, the type II cytokeratin exclusively expressed in the companion layer, was performed in 9 families. In 3 of these 9 families, a heterozygous glutamic acid and lysine mutation, E337K, was identified in the L2 linker region of K6HF. CONCLUSIONS: Diffuse partial woolly hair can be associated with loose anagen hair syndrome. A keratin mutation, E337K in K6HF, was possibly causative in 3 of the 9 families studied. Another keratin, and possibly the type I partner of K6hf, could be responsible for loose anagen hair syndrome in other patients, or the gene involved may be a minor gene.

HOXC13 is involved in the regulation of human hair keratin gene expression.

At present, HOXC13 is the only member of the HOX multigene family that produces a fragile hair phenotype when mutated or overexpressed in mice. To determine whether hair keratin genes are targets for this transcription factor, we analyzed the HOXC13 responsiveness of human hair keratin genes, whose expression matched that of nuclear HOXC13, immunologically revealed in cells of the lower hair-forming compartment of the human anagen hair follicle. We show that HOXC13, but not a homeobox-deleted HOXC13, strongly activated the promoters of the genes, with the respective proximal promoter regions being sufficient for optimal activation. The hair keratin promoters contained numerous putative Hox binding core motifs TAAT, TTAT, and TTAC. Electrophoretic mobility shift assays revealed that HOXC13 bound exclusively to distinct TAAT and TTAT core motifs that were clearly concentrated in the proximal promoter regions. A comparison of the sequences flanking HOXC13 binding and nonbinding core motifs, respectively, allowed the deduction of an extended 8-bp HOXC13 consensus binding sequence TT(A/T)ATNPuPu. Thus, the DNA binding conditions for HOXC13 were distinct from those of other members of the paralogous group 13, i.e. murine Hoxb13 and HOXd13, for which previous investigations yielded the consensus binding sequence TTTA(T/C)NPuPu. Collectively, our data speak for a direct involvement of HOXC13 in the control of hair keratin expression during early trichocyte differentiation.

[Endogenous synthesis of short-lived messenger-RNA-species in oocytes ofDysdercus intermedius Dist. after termination of vitellogenesis]. / Endogene Synthese kurzlebiger Messenger-RNS in der Oocyte vonDysdercus intermedius Dist. nach Abschluß der Vitellogenese.

Non-ribosomal RNA is synthesized in oocytes of the telotrophicmeroistic ovary ofDysdercus intermedius Dist. during late oogenesis, 4-14 h before they become mature eggs. RNA is labelled "in vivo" by radioactive RNA precursors from a nucleotide pool which is established in the ooplasm prior to chorion-formation.RNA synthesized by late oocytes is characterized by a high turnover rate and appears first as a high molecular weight precursor which is converted during a few hours to smaller non-ribosomal RNA species of 30-5 S. In newly-laid eggs RNA molecules synthesized by the oocyte are no longer present. Their degradation products can be found within the nucleotide fraction. Thus, in contrast to RNA synthesized by trophocytes and stored in eggs in a stable form, endogeneously-formed RNA is not conserved for use in embryogenesis.Endogeneous RNA of oocytes is found to have a high content of poly(A)-segments. Fifty-seven percent of these are hybridizable with poly(U) immobilised on glassfiber filters. A few hours before the maturation of eggs, shortlived RNA synthesized by the oocyte is found in association with polysomes. By incubation of such polysomes in an "in vitro" protein-synthesizing system, polypeptides are formed which show a characteristic banding pattern after separation on SDS-polyacrylamide gels. The apparent molecular weights for the 4 main proteins are 65.000, 48.000, 44.000, and 40.000. There is no evidence for identity of any one of these proteins with a chorion protein.Some of the heterogeneous population of RNA molecules synthesized by late oocytes are characterized by a short lifetime, a relatively high content of poly(A) and the ability to activate protein synthesis "in vivo" or "in vitro", thus suggesting a function like that of mRNA.

[Ribonucleoprotein-particles from telotrophic meroistic ovary ofDysdercus intermedius Dist. (Heteroptera, Pyrrhoc.) and their behaviour in a cell-free protein synthesizing system]. / Ribonucleoprotein-Partikel aus dem telotroph-meroistischen Ovar vonDysdercus intermedius Dist. (Heteroptera, Pyrrhoc.) und ihr Verhalten im zellfreien Proteinsynthesesystem.

In the telotrophic meroistic ovary ofDysdercus intermedius Dist. the oocyte is provided in a previtellogenic state with RNP-particles synthesized by trophocytes, transported in nutritive cords and deposited in oocytes. Ribosomes prepared by differential centrifugation from trophic tissue and mature eggs were fractionated into RNP-particle classes using a linear sucrose gradient. Particles were labeled by injecting radioactive RNA-precursors. Two hours later in trophic tissue besides 80 s monosomes and ribosomal subunits labeled RNP-particles are detected sedimenting slower than small ribosomal subunits (10-40 s). They are not transferred into the polyribosomal protein synthesizing complex of trophocytes. The ribosome fraction from eggs laid 8 days after injection of radioactive precursor contains the same labeled RNP-particle groups as the trophocytes. Because of the special character of telotrophic meroistic insect ovary (no detectable synthetic activity of oocyte nuclei during the growth phase, no contribution of high molecular weigth RNA from follicle cells to oocyte) these particles in the fresh laid bug-egg can be considered as products which are synthesized in trophocytes and therefore as depot-forms of maternal information for early embryonic protein synthesis.80 s monosomes contain exclusively rRNA (28 s and 18 s). In the slow sedimenting RNP-particles RNA was detected showing the following mRNA-characteristics: 1. a heterogenic molecular size with maximum at 7-9 s (proved by gelelectrophoretic analyses of radioactive labeled RNA isolated from particle groups), 2. a high content of Poly A-segments (proved by retention on nitrocellulose filters), 3. a stimulating capacity on amino acid incorporation in a cell-free protein synthesizing system.In a homologous cell-free system composed of bug components native mRNP-particles (10-40 s) from eggs and nurse cells cause an inhibition of amino acid incorporation into proteins. For this inhibitory effect proteins are responsible which dissociate from the particles in a medium of high ionic strength (0.5 M KCl). Under the same experimental conditions factors are dissociated from larval RNP-particles which show a stimulating effect on amino acid incorporation. Therefore inhibitory factors are thought to be structural or accessory specific components of maternal mRNP-particles. M-RNA from egg particles freed from inhibitory factors can be translated by specific factors which are detected-like the inhibitors in the ribosomal wash fraction.

[RNA-Synthesis in telotrophic meroistic ovarioles of dysdercus intermedius DIST. (heteroptera, pyrrhoc.)]. / Ribonucleinsäuresynthese in der telotroph-meroistischen Ovariole vonDysdercus intermedius Dist. (Heteroptera, Pyrrhoc.).

RNA in insect ovaries was investigated by polyacrylamide gel electrophoresis to study site of synthesis, transportation and incorporation in mature eggs. Telotrophic-meroistic ovarioles ofDysdercus intermedius were selected for this work since they could be dissected in distinct portions: the apical one with polyploid nurse cells and the vitellarium with oocytes covered by a follicular epithelium. RNA was labeled by injecting radioactive precursors or incubating isolated ovarioles in vitro.In nurse cells labeled rRNA, tRNA and non-ribosomal RNA were found and evidence was presented for the processing of a 39s rRNA precursor molecule in 2 steps (36s and 32s) into 28s rRNA while the 18s rRNA was directly derived from the 36s molecule. A low concentration (0.5 Μg/ml) of α-Amanitin inhibited synthesis of non-ribosomal RNA thereby revealing a more distinct processing pattern for rRNA, while a high concentration (5 Μg/ml) blocked processing at the 36s molecule and led to its accumulation. Appropriate concentrations of Actinomycin D inhibited rRNA synthesis while distinct peaks of non-ribosomal RNA became apparent.RNA synthesized in the follicular cells was similar to the RNA made in the nurse cells only in the high molecular weight RNA. Some material, tentatively named "nucleotide fraction", with a mobility higher than tRNA was detected and could be localized in yolk granules. It was separable from RNA by chromatography on Sephadex G 100 and had an absorption maximum at 255 run. The "nucleotide fraction" could be rapidly labeled. It seemed to be utilized in RNA synthesis during embryogenesis. Mature eggs contained a relatively high amount of the "nucleotide fraction". If a single injection with precursor was done 4 days before analysing the RNA of mature eggs most of the label was found in the "nucleotide fraction". Eggs collected 5-8 days after injection contained only high molecular labeled RNA. This observation correlated well with the timing of egg maturation. It takes 4 days for a group of oocytes to reach the distal part of the vitellarium where yolk production and maturation go on for another 4 days. It is apparent, therefore, that the follicular epithelium contributes the "nucleotide fraction" to the oocyte, while most of the other RNA, including a stable polydisperse class of RNA, is made in the nurse cells and is transported into the oocyte.