Molecular cloning of the mouse cell adhesion molecule uvomorulin: cDNA contains a B1-related sequence.

A clone (F20) containing coding sequences for the cell adhesion molecule uvomorulin was isolated by immunological techniques from cDNA library in the expression vector lambda gt11. The beta-galactosidase-uvomorulin fusion protein was used to affinity 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.

The primary structure of chicken B-creatine kinase and evidence for heterogeneity of its mRNA.

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.

Spatial and temporal patterns of Krüppel gene expression in early Drosophila embryos.

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.

Production of phenocopies by Krüppel antisense RNA injection into Drosophila embryos.

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.

Molecular genetics of Krüppel, a gene required for segmentation of the Drosophila embryo.

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.

Molecular cloning and expression during myogenesis of sequences coding for M-creatine kinase.

Sequences complementary to muscle poly(A)+RNA were cloned in the plasmid pBR322 and the resulting colonies were screened by colony hybridization with labeled cDNA derived from skeletal muscle and smooth muscle (gizzard). The skeletal muscle-specific clones were further screened by RNA blotting hybridization for a muscle mRNA having the size expected for a putative type M creatine kinase (M-CK) mRNA. The remaining clones with the expected hybridization properties were finally characterized by hybrid-selected translation, and a cloned sequence was shown to contain DNA hybridizing to mRNA that could be translated into M-CK. This plasmid, pMCK1, was further characterized by restriction mapping. Blot analysis of total cell RNA from differentiating myogenic cell cultures showed accumulation of M-CK mRNA in cultures older than 42 hr but not in young little-differentiated cultures.

Occurrence of heterogenous forms of the subunits of creatine kinase in various muscle and nonmuscle tissues and their behaviour during myogenesis.

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 creatine kinase and all gave the same results. In the course of myogenic 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 the 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.

The Mr 165,000 M-protein myomesin: a specific protein of cross-striated muscle cells.

The tissue specificity of chicken 165,000 M-protein, tentatively names "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 [35S]methionine into immunoprecipitable protein of differentiating cells increased sharply after approximately 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.