Establishment of the steroid-producing Leydig cell lineage is an event downstream of

D-type cyclins and cyclin E represent two very distinct classes of mammalian G1 cyclins. We have generated a mouse strain in which the coding sequences of the cyclin D1 gene (Ccnd1) have been deleted and replaced by those of human cyclin E (CCNE). In the tissues and cells of these mice, the expression pattern of human cyclin E faithfully reproduces that normally associated with mouse cyclin D1. The replacement of cyclin D1 with cyclin E rescues all phenotypic manifestations of cyclin D1 deficiency and restores normal development in cyclin D1-dependent tissues. Thus, cyclin E can functionally replace cyclin D1. Our analyses suggest that cyclin E is the major downstream target of cyclin D1.

The side chain in plant sterols can have either a methyl or ethyl addition at carbon 24 that is absent in cholesterol. The ethyl addition is the product of two sequential methyl additions. Arabidopsis contains three genes-sterol methyltransferase 1 ( SMT1) , SMT2, and SMT3-homologous to yeast ERG6 , which is known to encode an S -adenosylmethioninedependent C-24 SMT that catalyzes a single methyl addition. The SMT1 polypeptide is the most similar of these Arabidopsis homologs to yeast Erg6p. Moreover, expression of Arabidopsis SMT1 in erg6 restores SMT activity to the yeast mutant. The smt1 plants have pleiotropic defects: poor growth and fertility, sensitivity of the root to calcium, and a loss of proper embryo morphogenesis. smt1 has an altered sterol content: it accumulates cholesterol and has less C-24 alkylated sterols content. Escherichia coli extracts, obtained from a strain expressing the Arabidopsis SMT1 protein, can perform both the methyl and ethyl additions to appropriate sterol substrates, although with different kinetics. The fact that smt1 null mutants still produce alkylated sterols and that SMT1 can catalyze both alkylation steps shows that there is considerable overlap in the substrate specificity of enzymes in sterol biosynthesis. The availability of the SMT1 gene and mutant should permit the manipulation of phytosterol composition, which will help elucidate the role of sterols in animal nutrition.

The side chain in plant sterols can have either a methyl or ethyl addition at carbon 24 that is absent in cholesterol. The ethyl addition is the product of two sequential methyl additions. Arabidopsis contains three genes-sterol methyltransferase 1 (SMT1), SMT2, and SMT3-homologous to yeast ERG6, which is known to encode an S-adenosylmethionine-dependent C-24 SMT that catalyzes a single methyl addition. The SMT1 polypeptide is the most similar of these Arabidopsis homologs to yeast Erg6p. Moreover, expression of Arabidopsis SMT1 in erg6 restores SMT activity to the yeast mutant. The smt1 plants have pleiotropic defects: poor growth and fertility, sensitivity of the root to calcium, and a loss of proper embryo morphogenesis. smt1 has an altered sterol content: it accumulates cholesterol and has less C-24 alkylated sterols content. Escherichia coli extracts, obtained from a strain expressing the Arabidopsis SMT1 protein, can perform both the methyl and ethyl additions to appropriate sterol substrates, although with different kinetics. The fact that smt1 null mutants still produce alkylated sterols and that SMT1 can catalyze both alkylation steps shows that there is considerable overlap in the substrate specificity of enzymes in sterol biosynthesis. The availability of the SMT1 gene and mutant should permit the manipulation of phytosterol composition, which will help elucidate the role of sterols in animal nutrition.

Sciatic nerve axons in cyclin D1 knockout mice develop normally, become properly ensheathed by Schwann cells, and appear to function normally. However, in the Wallerian degeneration model of nerve injury, the mitotic response of Schwann cells is completely inhibited. The mitotic block is Schwann cell autonomous and developmentally regulated. Rescue analysis (by "knockin" of cyclin E) indicates that D1 protein, rather than regulatory elements of the D1 gene, provides the essential Schwann cell function. Genetic inhibition of the Schwann cell cycle shows that neuronal responses to nerve injury are surprisingly independent of Schwann cell mitotic responses. Even axonal regrowth into the distal zone of a nerve crush injury is not markedly impaired in cyclin D1-/- mice.

Cyclin E1 (formerly called cyclin E) and the recently described cyclin E2 belong to the family of E-type cyclins that operate during the G1͞S phase progression in mammalian cells. The two E-cyclins share a catalytic partner, cyclin-dependent kinase 2 (CDK2), and activate their associated kinase activities at similar times during cell cycle progression. Despite these similarities, it is unknown whether the two proteins perform distinct functions, or, alternatively, they control S-phase entry of different cell types in a tissue-specific fashion. To start addressing in vivo functions of E-cyclins, we determined the expression pattern of cyclins E1 and E2 during normal mouse development. We found that the two E-cyclins showed very similar patterns of expression; both were expressed within the proliferating compartment during embryo development. Analyses of cells and tissues lacking members of the retinoblastoma (pRB) family of proteins revealed that the expression of both cyclins is controlled in a pRB-dependent, but p107-and p130-independent fashion, likely through the pRB-dependent E2F transcription factors. We also found that cyclins E1 and E2 are expressed at high levels in mouse breast tumors driven by the Myc oncogene. Last, we found that cyclin E2 is overexpressed in Ϸ24% of analyzed human mammary carcinomas. Collectively these findings suggest that the expression of cyclins E1 and E2 is governed by similar molecular circuitry. Cyclins are key components of the core cell cycle machinery in mammalian cells. Two classes of cyclins operate during the G 1 phase: D-type cyclins and cyclin E (1).D-cyclins specify the family of three closely related proteins (cyclins D1, D2, and D3). The levels of D-cyclins are controlled by the extracellular environment. Once induced, D-cyclins associate with cyclin-dependent kinases CDK4 or CDK6 and phosphorylate the retinoblastoma tumor suppressor gene product (pRB). This phosphorylation leads to release of pRB-bound transcription factors, notably the E2F family of transactivators, and to derepression or activation of E2F-controlled genes, such as cyclin E. In addition, cyclin D-CDK complexes control the activity of cyclin E-CDK2 holoenzyme by titrating away the CDK inhibitors p27 Kip1 and p21Cip1 from cyclin E-CDK2 complexes to cyclin D-CDK4͞6 complexes, thereby triggering the kinase activity of cyclin E-CDK2 (1). Hence, D-type cyclins serve to couple the extracellular mitogenic stimulation with activation of cyclin E. While the three D-cyclins are biochemically indistinguishable, each of these cyclins shows a unique, tissue-specific pattern of expression during development and in adult tissues, and each is differentially controlled by distinct upstream activating pathways (2-5).Cyclin E is believed to control G 1 ͞S phase progression. It associates with CDK2 and activates its kinase activity shortly before entry of cells into the S phase (6, 7). The targets for cyclin E-CDK2 kinase are largely unknown. Cyclin E is believed to control S phase entry by phosphorylating proteins in...