No abstract

Krüppel is a member of the 'gap' class of segmentation genes of Drosophila melanogaster, mutations of which cause contiguous groups of segments of the fruitfly embryo to fail to develop. In the case of Krüppel mutant embryos, thoracic and anterior abdominal segments are deleted. The molecular cloning of the Krüppel locus will lead to an understanding of the crucial role that gap genes seem to have in early embryonic development. It has already allowed the identification of a blastoderm-specific Krüppel transcript and the phenotypic rescue of mutant embryos by injected cloned DNA.

The Krüppel (Kr) locus is a member of the 'gap' class of segmentation genes of Drosophila melanogaster. Mutations at the Kr locus cause the deletion of contiguous segments from the embryonic body pattern. We have elucidated the spatial and temporal characteristics of Kr gene expression during early embryo development, the localization of cytoplasmic Kr+ activity and its spatial requirement for normal segmentation.

The demonstration that a specific messenger RNA can be functionally inactivated in vivo by hybridization to complementary polynucleotide sequences suggests a direct approach to the study of gene function in cells of higher organisms. The experiments described here were designed to inhibit, by complementary RNA sequences, a specific gene function affecting the fate of the Drosophila embryo. We used the SP6 vector in vitro transcription system to transcribe parts of the normally untranscribed (nonsense) strand of the Krüppel (Kr) gene into complementary Kr RNA (Kr antisense RNA). Wild-type Drosophila embryos, injected with this RNA, developed into phenocopies of Kr mutant embryos.

The tissue specificity of chicken 165,000 M-protein, tentatively named "myomesin," a tightly bound component of the M-line region of adult skeletal and heart myofibrils, was investigated by immunological techniques . Besides skeletal and heart muscle, only thymus (known to contain myogenic cells) was found to contain myomesin . No myomesin could, however, be detected in smooth muscle or any other tissue tested . This result was confirmed in vitro on several cultured embryonic cell types. Only skeletal and heart muscle cells, but not smooth muscle or fibroblast cells, showed the presence of myomesin . When the occurrence and the distribution of myomesin during differentiation of breast muscle cells in culture were studied by the indirect immunofluorescence technique, this protein was first detected in postmitotic, nonproliferating myoblasts in a regular pattern of fluorescent cross-striations . In electron micrographs of sections through young myotubes, it could be shown to be present within the forming H-zones of nascent myofibrils . In large myotubes the typical striation pattern in the M-line region of the myofibrils was observed . Synthesis of myomesin measured by incorporation of [31S] methionine into immunoprecipitable protein of differentiating cells increased sharply after -48 h in culture, i .e ., at the time when the major myofibrillar proteins are accumulated . No significant amounts of myomesin were, however, found in cells prevented from undergoing normal myogenesis by 5'-bromodeoxyuridine .The results indicate that myomesin (a) is a myofibrillar protein specific for cross-striated muscle, (b) represents a highly specific marker for cross-striated muscle cell differentiation, and (c) might play an important role in myofibril assembly and/or maintenance.The temporal appearance of myofibrillar proteins as well as the composition and organization of myofibrils in differentiating muscle cells have been subjects of intense studies in recent years (for review, see references 1, 5, 11, 13, 17, 19, 24, and 30) . Investigations of the properties of a number ofnewly detected, so-called minor components of the myofibrillar compartment (12,14,25,28,31,48,53) have provided a better insight into the molecular organization of the unit of contractility, the sarcomere . There is, however, still considerable lack of knowledge regarding the sequence of appearance and organization of the myofibrillar structures during development (1,13,19,37,49). One of the major transverse structures in a mature cross-striated muscle fiber, the Z-band, has been described as the initial site of myofibrillogenesis (19), whereas the second one, the so-called M-line, has only recently been found to play a role in the organization of the assembly of the myofibrillar structure (10,12,16,43 Our own investigations on the structure and function of the M-line region ofchicken skeletal and heart muscle indicate the presence of two major protein components: the muscle type isoprotein of creatine kinase (MM-CK) (48, 50, 52) and the 165...

cDNA clones for chicken B-CK were isolated by immunoscreening from a gizzard cDNA library constructed in the expression vector lambda gtll. The entire coding portion in addition to the complete 3' untranslated region and 42 bp of the 5' noncoding part are represented in the clone H4. On RNA blots H4 insert DNA hybridized to a 1600 bp poly(A)+ RNA from gizzard, brain and heart but not to breast or skeletal muscle RNA. In vitro generated sense strand transcripts of H4 insert DNA were translated in vitro into a protein indistinguishable from isolated, authentic B-CK. The distinct nucleotide sequences of H4 insert DNA and M-CK cDNA were translated into 82% homologous amino acid sequences. Sequence heterogeneity among the B-CK cDNA clones within both the 3' noncoding and even in the coding region indicates the existence of multiple B-CK mRNA species.

A clone (F20) containing coding sequences for the cell adhesion molecule uvomorulin was isolated by immunological techniques from a cDNA library in the expression vector Agtll.' The ,B-galactosidase-uvomorulin fusion protein was used to affiity purify anti-uvomorulin antibodies. Affinity-purified antibodies recognized uvomorulin from cell lysates of embryonal carcinoma cells and reacted with the cell surface of embryonal carcinoma cells. The 1.8-kilobase cDNA insert hybridized to a single 4.3-kilobase poly(A)+ RNA species found only in cells expressing uvomorulin. Part of the nontranslated 3' sequences of the cloned uvomorulin cDNA is homologous to the interspersed B1 repeat of the mouse genome.Cell-cell interactions during embryonic development and in tissue organization involve specific cell-adhesion molecules (CAM) that are functionally defined by antibodies that interfere with cell-cell adhesion (1-5).Uvomorulin is involved in the compaction process of mouse morulae (6, 7). Later in development uvomorulin is expressed on epithelial cells independent of their germ-layer origin (8, 9)'. In adult tissue, the localization of uvomorulin is restricted to the intermediate junctions of intestinal epithelial cells (10). Anti-uvomorulin Fab fragments block the aggregation of primary mouse embryonic liver cells (11), and the rat anti-uvomorulin monoclonal antibody DECMA-1 disrupts the monolayer of canine kidney epithelial (MDCK) cells (34) indicating that uvomorulin also serves as an adhesive factor on adult cells.Uvomorulin has been characterized as a 120-kDa cell surface glycoprotein (gpl20) from which an 84-kDa fragment (gp84) can be released by trypsin digestion in the presence of Ca2l (7). Antibodies prepared against each of these two proteins recognize two additional proteins with apparent molecular sizes of 102 (plO2) and 92 (gp92) kDa (8, 9), respectively. 'The relation of these proteins to gpl20 is beginning to be understood (11), and it is clear that at least p102 is a true cellular component also expressed on the cell surface (D.V. and R.K., unpublished results). Molecular studies on uvomorulin require molecular probes to establish the relation of the different proteins that share common antigenic sites and a possible homology of uvomorulin with other CAM.The present paper describes the molecular cloning of uvomorulin cDNA and the uvomorulin mRNA and gives an initial characterization of part of the uvomorulin gene.MATERIALS AND METHODS Cells. The following cells were used: embryonal carcinoma (EC) cells [PCC4azal, F9 (12,13), and 3/A/1-Dl-an EC cell-derived embryonic fibroblast cell line (14)] and NIH/3T3 cells.Antibodies. The following rabbit anti-uvomorulin sera were used: anti-gpl20 and anti-gp84 (9) and anti-p102 (D.V. and R.K., unpublished data). A rat monoclonal antibody (DECMA-1) has been selected against gp84 (34). This monoclonal antibody blocks cell adhesion in a functional cell aggregation assay and recognizes the known set of uvomorulin proteins in immunoprecipitation and immunoblot exper...

Purified, homodimeric creatine kinases from chicken were subjected to two-dimensional gel analysis under dissociating conditions. Each of the subunits M-creatine kinase and B-creatine kinase was resolved into a basic and an acidic subspecies with very similar mobilities in the sodium dodecylsulfate dimension. The M-creatine kinase subspecies were found in myogenic cells, fast muscle, slow muscle and the B-creatine kinase subspecies were present in heart, gizzard and brain. The creatine kinase subunits were identified in these tissues by a variety of methods like immunoreplicas of two-dimensional gels, immunoprecipitations, or coelectrophoresis with purified creatinc kinasc and all gave the same results. In the course of myogcnic development in vitro the subspecies were synthesized coordinately and no indication was found for a differential regulation of any of the subspecies of the creatine kinase subunits. No radioactive phosphorus was incorporated into either one of thc subspecies, hence phosphorylation could be ruled out as the source of heterogeneity. Furthermore, peptide mapping analysis of partial proteolytic digests did not reveal differences among the subspecies of the same subunit. Not only chicken but also rat creatine kinase displayed this type of heterogeneity. All subspecies were observed after translation of chicken RNA in a cell-free protein-synthesizing system. The heterogeneity probably might best be explained by the existence of multiple, but closely related genes for the creatine kinase subunits.Creatine kinase is a dimeric enzyme that plays a crucial role in the energy metabolism. It is especially characteristic of muscle tissue, but also occurs in other tissues [1,2]. The cytoplasmic nonmitochondrial enzymes are made up from two different kinds of subunits with a M , of 40000, the M-creatine kinase subunit and the B-creatine kinase subunit. MM-creatine kinase is found in appreciable amounts only in adult muscle, whereas BB-creatine kinase is more widely distributed in smooth muscle, chicken heart, brain and a variety of other tissues. During the differentiation of chicken myogenic cells in cultures as well as in embryonic muscle, an isoenzyme transition is observed from BB-creatine kinase to MM-creatine kinase, the form typical for the differentiated state. During this transition the subunits combine at random to yield homodimers and also active heterodimers MBcreatine kinase [3 -51. The subunits forming the active isoenzymes are very likely the products of at least two different genes as indicated by the differences of amino acid composition [6], the lack of immunological cross-reactivity [4,6,7] and differences in the metabolism of the corresponding mRNAs coding for thc two different subunits [S]. In addition M-creatine kinase is found in the M-line of adult skeletal myofibrils as well as in the myofibrils of cultured cells, suggesting a specific property of the MM-creatine kinase dimer whichAhhreviutions. M-creatine kinase and B-creatine kinase. subunit types of creatine kinase; MM-creati...